Multiple virus resistance in plants

ABSTRACT

The present invention provides gene targets, constructs and methods for the genetic control of plant disease caused by multiple plant viruses. The present invention relates to achieving a plant protective effect through the identification of target coding sequences and the use of recombinant DNA technologies for post-transcriptionally repressing or inhibiting expression of the target coding sequences of plant-parasitic viruses. Protein-expression based approaches may also be utilized to augment phenotype resistance. Thus, transcription of a single transgenic event comprising one or more plant expression cassettes can allow for broad spectrum resistance of a plant to multiple plant viral strains and species among the geminiviruses, tospoviruses, and potexviruses.

This application is a continuation of U.S. patent application Ser. No.12/763,790, filed Apr. 20, 2010, which application claims the benefit ofpriority of U.S. Provisional Application Ser. No. 61/171,021, filed Apr.20, 2009, the entire disclosures of which are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods and compositions forenhancing resistance to multiple plant viruses.

2. Description of Related Art

Solanaceous plants are subject to multiple potential disease causingagents, including virus-induced diseases that are responsible for majorcrop losses worldwide. For many RNA viruses, expression of transgeniccoat protein (CP) or replicase blocks the progression of the virusinfectious process. RNA-based resistance makes use of the plantpost-transcriptional gene silencing (PTGS) mechanism to degrade viralRNAs. However, such approaches may yield resistance that is narrowlybased and/or not durable, especially with rapidly spreading/evolving newviral species or isolates. In some instances, classically-defined(non-transgenic) resistance traits are available to aid in developmentof virus resistant plants. Additionally, control of plant pests, such asinsects that serve to transmit plant viruses, may help to limit lossesdue to viral infection of plants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic diagram of genome organization of viruses of interest.

FIG. 2A: Schematic diagram illustrating approach for identifyingsequence efficacious for plant virus control.

FIG. 2B: Schematic diagram of a typical begomovirus DNA-A genome showinglocation of regions screened for effectiveness for viral control whenexpressed as inverted repeats. Numbered gray arrows represent portionsof genome that were tested.

FIG. 2C: Schematic diagram of a typical potexvirus (Pepino mosaic virus;PepMV) genome showing location of regions screened for effectiveness forviral control when expressed as inverted repeats. Numbered gray arrowsrepresent portions of genome that were tested.

FIG. 2D: Schematic diagram of a tospovirus (e.g. Tomato spotted wiltvirus (TSWV) genome showing location of regions screened foreffectiveness for viral control when expressed as inverted repeats.Numbered gray arrows represent portions of genome that were tested.

FIG. 3: Virus resistance correlating with siRNA production intransformed tomato plants.

FIG. 4: Exemplary artificial dsRNA fusion constructs for conferringmultiple virus resistance (“MVR”).

FIG. 5: Suitable 21 nt sequences (among SEQ ID NOs:1-42) that wereanalyzed against five targeted Geminiviruses (perfect match: doubleunderline; G:U mis-match: single underline; other mis-match or notutilized: not underlined).

FIG. 6: Suitable 21 nt sequences (among SEQ ID NOs:43-70) that wereanalyzed against tospoviruses (perfect match: double underline; G:Umis-match: single underline; other mis-match or not utilized: notunderlined).

FIG. 7A, 7B: Suitable 21 nt sequences (among SEQ ID NOs:71-154) thatwere analyzed against targeted potexviruses (perfect match: doubleunderline; G:U mis-match: single underline; other mis-match or notutilized: not underlined).

FIG. 8: Schematic of exemplary construct for deploying multipleengineered miRNAs in one transgenic cassette, such as with phasedsiRNAs.

FIG. 9: Schematic diagram illustrating expression cassette for deployingmultiple modes of action for virus resistance.

FIG. 10: Additional exemplary constructs for deploying multipleengineered miRNAs in a transgenic cassette, as well as for expressingmiRNA along with dsRNA.

FIG. 11A, 11B: Scanning of regions of the tospovirus genome to definesegments which may be expressed as dsRNA with anti-viral efficacy. Xaxis represents individual events and target regions (CP: coat protein;GP: envelope glycoprotein; and RdRP: RNA-dependent RNA polymerase). Yaxis represents % of transgenic R₁ plants displaying virus resistance.FIG. 11A: TSWV results; FIG. 11B: CaCV and GBNV results. “CP4+” refersto presence of the selectable marker gene linked to the dsRNA-encodingsequence, in R₁ plants.

FIG. 12: Regions of the PepMV genome tested for effectiveness ingenerating dsRNA-mediated resistance against this potexvirus (CP: coatprotein; Mov: movement protein; RdRP, or RdR: RNA-dependent RNApolymerase; TGB: Triple gene block protein).

FIG. 13A, 13B: Regions of geminivirus genome assayed for effectivenessin generating dsRNA-mediated resistance against this virus group. (CP:coat protein; Rep: replication protein). Other transgenic plants containa glyphosate resistance gene. “0%-100%” denotes the percentage of R₁plants that are selectable-marker positive and virus resistant. FIG.13A: representative results for TYLCV and ToSLCV; FIG. 13B:representative results for PepGMV, PHYVV, and ToLCNDV. For PHYVV andToLCNDV events, “Spc” refers to the presence of a selectable marker geneconferring spectinomycin resistance. Other events were transformed witha construct comprising a selectable marker gene conferring glyphosateresistance (“CP4 positive”).

FIG. 14: Schematic diagram illustrating representative expressioncassettes for targeting of multiple viruses in multiple virus families.

FIG. 15: Results of using an artificial dsRNA fusion construct targetingCP expression of tospoviruses and PepMV (potexvirus), for multiple virusresistance as discussed in Example 3. Construct used is schematicallyshown in FIG. 4B, bottom construct: TSWV (160 bp), TSWV (296 bp), PepMV(231 bp), CaCV/GBNV (232 bp).

FIG. 16: Depicts resistance observed against inoculated CaCV ininoculated CP4 positive R₁ plants transformed with a constructcomprising tospovirus terminal repeat sequences (SEQ ID NOs:167, 168,376, 377, 378, as found in SEQ ID NO:455).

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for obtainingplants resistant to multiple plant viruses. In one aspect, the presentinvention provides a tomato plant comprising resistance to a pluralityof plant virus species. In certain embodiments, the resistance isprovided by at least two different modes of action selected from thegroup consisting of dsRNA, miRNA, and inhibition of virion assembly. Inother embodiments, the resistance is provided by at least threedifferent modes of action. The resistance of the tomato plant maycomprise resistance against a begomovirus, tospovirus or potexvirus.

In certain embodiments, the resistance provided to at least one of theplant virus species is provided by expression of a nucleic acidconstruct that produces dsRNA. In some embodiments the resistanceprovided to at least one of the plant virus species is provided byexpression of a dsRNA fusion construct. In some embodiments of theinvention, the dsRNA interferes with expression of a virus coat proteingene, a virus movement protein gene or a virus replication gene. Inparticular embodiments, the nucleic acid construct which produces dsRNAcomprises a sequence selected from the group consisting of SEQ IDNOs:379-455.

In other embodiments, the resistance provided to at least one of theplant virus species is provided by expression of a nucleic acidconstruct that produces miRNA. Thus, in certain embodiments, theresistance against a begomovirus or tospovirus is provided by a sequenceencoded by a stacked miRNA expression cassette. In yet otherembodiments, the miRNA interferes with expression of a virus coatprotein gene, a virus movement protein gene or a virus replication gene.In particular embodiments, the miRNA comprises a sequence selected fromthe group consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51,55, 59, 63, 67, 71, 85, 99, 113, 127, and 141.

In certain embodiments, the tomato plant comprises (a) resistanceagainst a begomovirus which is provided by expression of dsRNA whichinterferes with expression of a begomovirus replication gene; (b)resistance against a tospovirus or potexvirus which is provided byexpression of a dsRNA which interferes with expression of a virus coatprotein gene or virus movement protein gene; (c) resistance against apotexvirus which is provided by expression of a nucleic acid constructwhich produces miRNA; or (d) resistance against a begomovirus ortospovirus which is provided by a sequence encoded by a stacked miRNAexpression cassette.

In other embodiments, a tomato plant is provided wherein resistanceprovided to at least one of the plant virus species is provided byexpression of a tospovirus genome segment terminal sequence thatinhibits virion assembly. In certain embodiments, resistance provided toat least one of the plant virus species is provided by inhibiting virionassembly, wherein virion assembly is inhibited by a sequence comprisedwithin a nucleic acid construct comprising a first nucleic acid segmentand a second nucleic acid segment, wherein the first and second segmentsare substantially inverted repeats of each other and are linked togetherby a third nucleic acid segment, and wherein the third segment comprisesat least one terminal sequence of a tospovirus genome segment thatinhibits virion assembly. In particular embodiments, the third nucleicacid comprises a tospovirus genome terminal sequence selected from thegroup consisting of: a terminal sequence of a CaCV or GBNV L genomesegment, a terminal sequence of a CaCV or GBNV M genome segment, aterminal sequence of a CaCV or GBNV S genome segment, a tospovirusgenome terminal repeat sequence, a nucleic acid sequence comprising SEQID NO: 167, a nucleic acid sequence comprising SEQ ID NO:168, a nucleicacid sequence comprising SEQ ID NO:376, a nucleic acid sequencecomprising SEQ ID NO:377, a nucleic acid sequence comprising SEQ IDNO:378, and a nucleic acid sequence comprising SEQ ID NO: 455.

In certain embodiments, the tomato plant comprises resistance to virusesof at least two of the Geminiviridae, Bunyaviridae and Flexiviridaefamilies. Thus, in some embodiments the viruses are selected from thegenera potexvirus, tospovirus, and begomovirus. In particularembodiments, the viruses are selected from the group consisting of: a)at least one of TYLCV, ToSLCV, ToLCNDV, PHYVV, PepGMV; b) one or more ofTSWV, GBNV, CaCV; and c) PepMV. In a more particular embodiment, thepotexvirus is Pepino mosaic virus. In certain embodiments, thebegomovirus is TYLCV, ToLCNDV, PHYVV, ToSLCV, or PepGMV. In someembodiments, the tospovirus is CaCV, GBNV, or TSWV. In particularembodiments, the begomovirus is TYLCV and the potexvirus is Pepinomosaic virus; or the tospovirus is TSWV and the potexvirus is Pepinomosaic virus; or the wherein the begomovirus is TYLCV, the potexvirus isPepino mosaic virus, and the tospovirus is TSWV.

In some embodiments, the tomato plant may comprise a sequence selectedfrom the group consisting of SEQ ID NOs:156, 158, 160, 162, 164, 166,and 363-375. In those or other embodiments, the tomato plant comprises,or further comprises, a sequence selected from the group consisting of:SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85,99, 113, 127, 141, and 379-454. Thus, a tomato plant of the inventionmay comprise: (a) at least one sequence selected from the groupconsisting of SEQ ID NOs:379-454 and at least one sequence selected fromthe group consisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455; (b)at least one sequence selected from the group consisting of SEQ IDNOs:379-454 and at least one sequence selected from the group consistingof SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71,85, 99, 113, 127, and 141; or (c) at least one sequence selected fromthe group consisting of SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51,55, 59, 63, 67, 71, 85, 99, 113, 127, and 141 and at least one sequenceselected from the group consisting of SEQ ID NOs:167, 168, 376, 377,378, and 455.

In yet other embodiments, the tomato plant comprises at least oneheterologous nucleic acid sequence that confers viral resistanceselected from the group consisting of a) a nucleic acid sequence thatencodes an RNA sequence that is complementary to all or a part of afirst target gene; b) a nucleic acid sequence that comprises multiplecopies of at least one anti-sense DNA segment that is anti-sense to atleast one segment of said at least one first target gene; c) a nucleicacid sequence that comprises a sense DNA segment from at least onetarget gene; d) a nucleic acid sequence that comprises multiple copiesof at least one sense DNA segment of a target gene; e) a nucleic acidsequence that transcribes to RNA for suppressing a target gene byforming double-stranded RNA and that comprises at least one segment thatis anti-sense to all or a portion of the target gene and at least onesense DNA segment that comprises a segment of said target gene; f) anucleic acid sequence that transcribes to RNA for suppressing a targetgene by forming a single double-stranded RNA that comprises multipleserial anti-sense DNA segments that are anti-sense to at least onesegment of the target gene and multiple serial sense DNA segments thatcomprise at least one segment of said target gene; g) a nucleic acidsequence that transcribes to RNA for suppressing a target gene byforming multiple double strands of RNA and comprises multiple segmentsthat are anti-sense to at least one segment of said target gene andmultiple sense DNA segments of the target gene, and wherein saidmultiple anti-sense DNA segments and said multiple sense DNA segmentsare arranged in a series of inverted repeats; h) a nucleic acid sequencethat comprises nucleotides derived from a plant miRNA; and i) a nucleicacid sequence encoding at least one tospovirus terminal sequence thatinterferes with virion assembly. The invention also provides a plantwherein expression of the at least one heterologous nucleic acidsequence results in resistance to two or more viruses selected from thegroup consisting of: tospoviruses, begomoviruses, and potexviruses. Theplant may also further comprise a non-transgenic plant virus resistancetrait.

In another aspect of the invention, a transgenic seed is provided, ofany generation of the tomato plant comprising resistance to a pluralityof plant virus species, wherein the resistance is provided by at leasttwo different modes of action selected from the group consisting ofdsRNA, miRNA, and inhibition of virion assembly.

In yet another aspect, the invention provides a method for conferringresistance in a tomato plant to a plurality of plant virus species, themethod comprising expressing in the plant at least two nucleic acidsequences that collectively provide resistance to said plurality ofplant virus species, wherein at least 2 different modes of action areutilized to provide such resistance, comprising expression of at leasttwo sequences selected from the group consisting of: dsRNA, miRNA, and asequence which interferes with virion assembly. In certain embodiments,the resistance comprises resistance against a begomovirus, tospovirus orpotexvirus.

The resistance may be provided to at least one of the plant virusspecies by expression of a nucleic acid construct that produces dsRNA.In particular embodiments, resistance provided to at least one of theplant virus species is provided by expression of a dsRNA fusionconstruct. In more particular embodiments, the dsRNA interferes withexpression of a virus coat protein gene, a virus movement protein geneor a virus replication gene. In yet more particular embodiments, thenucleic acid construct comprises a sequence selected from the groupconsisting of SEQ ID NOs:379-455.

In other embodiments, resistance provided to at least one of the plantvirus species is provided by expression of a nucleic acid construct thatproduces miRNA. In one embodiment, it is contemplated that resistanceagainst a begomovirus or tospovirus is provided by a sequence encoded bya stacked miRNA expression cassette. The produced miRNA may furtherinterfere with expression of a virus coat protein gene, a virus movementprotein gene or a virus replication gene. In particular embodiments, themiRNA comprises a sequence selected from the group consisting of SEQ IDNOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99,113, 127, and 141.

Thus, in certain embodiments, (a) resistance against a begomovirus isprovided by expression of dsRNA which interferes with expression of abegomovirus replication gene; (b) resistance against a tospovirus orpotexvirus is provided by expression of a dsRNA which interferes withexpression of a virus coat protein gene or virus movement protein gene;(c) resistance against a potexvirus is provided by expression of anucleic acid construct which produces miRNA; or (d) resistance against abegomovirus or tospovirus is provided by a sequence encoded by a stackedmiRNA expression cassette.

In some embodiments resistance provided to at least one of the plantvirus species is provided by expression of a tospovirus genome segmentterminal sequence that inhibits virion assembly. In certain embodiments,resistance is provided to at least one of said plant virus species byinhibiting virion assembly, wherein virion assembly is inhibited by asequence comprised within a nucleic acid construct comprising a firstnucleic acid segment and a second nucleic acid segment, wherein thefirst and second segments are substantially inverted repeats of eachother and are linked together by a third nucleic acid segment, andwherein the third segment comprises at least one terminal sequence of atospovirus genome segment, expression of which inhibits virion assembly.Further, in particular embodiments, the third nucleic acid may comprisea tospovirus genome terminal sequence selected from the group consistingof: a terminal sequence of a CaCV or GBNV L genome segment, a terminalsequence of a CaCV or GBNV M genome segment, a terminal sequence of aCaCV or GBNV S genome segment, and a tospovirus genome terminal repeatsequence. In more particular embodiments, the terminal sequence orterminal repeat sequence comprises SEQ ID NO:167, SEQ ID NO:168, SEQ IDNO:376, SEQ ID NO:377, or SEQ ID NO: 378.

In other embodiments of the invention, the plurality of plant virusspecies are selected from at least two of the Geminiviridae,Bunyaviridae and Flexiviridae families. Thus, the viruses may beselected from the genera Potexvirus, Tospovirus, and Begomovirus. Incertain embodiments, the viruses are selected from the group consistingof: a) one or more of TYLCV, ToSLCV, ToLCNDV, PHYVV, PepGMV; b) one ormore of TSWV, GBNV, CaCV; and c) PepMV.

In other embodiments, the nucleic acid sequence comprises at least onegene suppression element for suppressing at least one first target gene.For instance, the method may comprise expressing in the plant: a) atleast one sequence selected from the group consisting of SEQ IDNOs:379-454 and at least one sequence selected from the group consistingof SEQ ID NOs:167, 168, 376, 377, 378, and 455; (b) at least onesequence selected from the group consisting of SEQ ID NOs:379-454 and atleast one sequence selected from the group consisting of SEQ ID NOs:1,7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127,and 141; or (c) at least one sequence selected from the group consistingof SEQ ID NOs:1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71,85, 99, 113, 127, and 141 and at least one sequence selected from thegroup consisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455.

Another aspect of the invention provides a transgenic cell of the tomatoplant comprising resistance to a plurality of plant virus species,wherein the resistance is provided by at least two different modes ofaction selected from the group consisting of dsRNA, miRNA, andinhibition of virion assembly.

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. The use of the term “or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only orthe alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and to “and/or.”When not used in conjunction closed wording in the claims orspecifically noted otherwise, the words “a” and “an” denote “one ormore.” The term “conferred by a transgene,” for example, thusencompasses one ore more transgene(s).

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps. Similarly, any plantthat “comprises,” “has” or “includes” one or more traits is not limitedto possessing only those one or more traits and covers other unlistedtraits.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and any specificexamples provided, while indicating specific embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

The following is a detailed description of the invention provided to aidthose skilled in the art in practicing the present invention. Those ofordinary skill in the art may make modifications and variations in theembodiments described herein without departing from the spirit or scopeof the present invention.

The present invention provides methods and compositions for geneticcontrol of virus diseases in plants, including Solanaceous plants suchas tomato (i.e. Lycopersicon or Solanum sp.), pepper (i.e. Capsicumsp.), petunia (i.e. Petunia sp.), and potato and eggplant (i.e. Solanumsp). In one embodiment RNA-mediated gene suppression can be conferred bythe expression of an inverted-repeat transgene cassette that generates apopulation of small interfering RNAs (siRNAs) derived from the dsRNAregion of a transgene transcript. Another RNA-mediated approach for genesuppression is by expression of one or more miRNA segments that “target”specific transcripts and lead to their degradation. Thus approachesincluding engineering dsRNA, miRNA, ta-siRNA and/or phased siRNA may beutilized in accordance with the invention. For instance,begomovirus-derived, or other virus-derived sequences targetingreplication, coat protein, and C2 and/or C3 proteins may also beutilized. Likewise, for control of potexviruses such as Pepino mosaicvirus, sequences targeting portions of coat (capsid) protein (“CP”),replication protein such as RNA-dependent RNA polymerase (“RdRP”),and/or one or more movement protein(s) (“MP”) which may include a triplegene block (“TGB”) or a “30K” MP may be used. For control oftospoviruses, sequences targeting, for instance, the coat protein (“CP”,also termed the nucleocapsid, “N” protein), RdRP, movement protein(“NsM”), and/or non-structural glyocoprotein(s) (encoded by “G1” or G2″genes) may similarly be utilized. Such sequences may correspond exactlyto sequences from one or more viral isolates, or may be variants, forinstance designed to increase their antiviral efficacy, or avoidnon-target effects.

In certain embodiments, multiple virus resistance (“MVR”) is achieved byutilizing dsRNA and/or miRNA expressed from a single transformedconstruct, or more than one construct. Further, a single construct maycomprise one or more expression cassettes that produce dsRNA and/ormiRNA that targets one or more functions necessary for plant viralinfection, multiplication, and/or transmission, as well as, in certainembodiments, one or more expression cassettes that produce at least onemRNA that encodes a protein, or portion of a protein, being targeted.Thus, resistance to multiple plant viruses may be achieved in a singletransgenic “event.” RNA mediated resistance may further be enhanced byprotein based approaches utilizing aptamer(s) that inhibit replication,expression or mutation in replicase or replication associated proteins,ssDNA binding proteins such as m13-G5 (e.g. U.S. Pat. No. 6,852,907;Padidam et al., 1999), for geminivirus resistance, or a peptide aptamerthat interferes with geminivirus replication may also be employed (e.g.Lopez-Ochoa et al., 2006).

It is also contemplated that inhibition of virion assembly, for instanceby a nucleic-acid based approach, may be utilized as a mode of action inproviding virus resistance to a tomato plant. This inhibition of virionassembly may be provided, for instance, by use of a tospovirus terminalsequence, such as a terminal repeat sequence. By “inhibition of virionassembly” is meant interference with the interaction between viralcapsid proteins and nucleic acid(s) which together may form a viralparticle (“virion”). Such interference may occur, for instance, byexpression of a sequence that can serve as an artificial substratecompeting for reverse transcriptase, and/or may occur by interferencewith proper circularization of replicating viral genome components.

Additionally, classical genetic resistance loci for tolerance in tomato,peppers, and other Solanaceous plants may be utilized, for instancethrough classical breeding approaches. In certain embodiments,protein-based approaches using tospovirus “N” gene (nucleocapsid; coatprotein) plus/minus inverted repeats and a potexvirus (e.g. Pepinomosaic virus) coat protein (CP) and replicase for resistance, are alsoprovided.

Methods of gene suppression may include use of anti-sense,co-suppression, and RNA interference. Anti-sense gene suppression inplants is described by Shewmaker et al. in U.S. Pat. Nos. 5,107,065,5,453,566, and 5,759,829. Gene suppression in bacteria using DNA whichis complementary to mRNA encoding the gene to be suppressed is disclosedby Inouye et al. in U.S. Pat. Nos. 5,190,931, 5,208,149, and 5,272,065.RNA interference or RNA-mediated gene suppression has been described by,e.g., Redenbaugh et al., 1992; Chuang et al., 2000; and Wesley et al.,2001.

Several cellular pathways involved in RNA-mediated gene suppression havebeen described, each distinguished by a characteristic pathway andspecific components. See, for example, reviews by Brodersen and Voinnet(2006), and Tomari and Zamore (2005). The siRNA pathway involves thenon-phased cleavage of a double-stranded RNA to small interfering RNAs(“siRNAs”). The microRNA pathway involves microRNAs (“miRNAs”),non-protein coding RNAs generally of between about 19 to about 25nucleotides (commonly about 20-24 nucleotides in plants) that guidecleavage in trans of target transcripts, negatively regulating theexpression of genes involved in various regulation and developmentpathways; see Ambros et al. (2003). Plant miRNAs have been defined by aset of characteristics including a paired stem-loop precursor that isprocessed by DCL1 to a single specific ˜21-nucleotide miRNA, expressionof a single pair of miRNA and miRNA* species from the double-strandedRNA precursor with two-nucleotide 3′ overhangs, and silencing ofspecific targets in trans (see Bartel (2004); Kim (2005); Jones-Rhoadeset al. (2006); Ambros et al. (2003)). In the trans-acting siRNA(“ta-siRNA”) pathway, miRNAs serve to guide in-phase processing of siRNAprimary transcripts in a process that requires an RNA-dependent RNApolymerase for production of a double-stranded RNA precursor;trans-acting siRNAs are defined by lack of secondary structure, a miRNAtarget site that initiates production of double-stranded RNA,requirements of DCL4 and an RNA-dependent RNA polymerase (RDR6), andproduction of multiple perfectly phased ˜21-nt small RNAs with perfectlymatched duplexes with 2-nucleotide 3′ overhangs (see Allen et al.,2005).

Many microRNA genes (MIR genes) have been identified and made publiclyavailable in a database (“miRBase”, available online atwww.microrna.sanger.ac.uk/sequences; also see Griffiths-Jones et al.(2003)). Additional MIR genes and mature miRNAs are also described inU.S. Patent Application Publications 2005/0120415 and 2005/144669, whichare incorporated by reference herein. MIR gene families appear to besubstantial, estimated to account for 1% of at least some genomes andcapable of influencing or regulating expression of about a third of allgenes (see, for example, Tomari et al. (2005); Tang (2005); and Kim(2005)). MIR genes have been reported to occur in intergenic regions,both isolated and in clusters in the genome, but can also be locatedentirely or partially within introns of other genes (both protein-codingand non-protein-coding). For a recent review of miRNA biogenesis, seeKim (2005). Transcription of MIR genes can be, at least in some cases,under control of a MIR gene's own promoter. The primary transcript,termed a “pri-miRNA”, can be quite large (several kilobases) and can bepolycistronic, containing one or more pre-miRNAs (fold-back structurescontaining a stem-loop arrangement that is processed to the maturemiRNA) as well as the usual 5′ “cap” and polyadenylated tail of an mRNA.See, for example, FIG. 1 in Kim (2005).

A “phased small RNA locus,” which transcribes to an RNA transcriptforming a single foldback structure that is cleaved in phase in vivointo multiple small double-stranded RNAs (termed “phased small RNAs”)capable of suppressing a target gene may also be employed (e.g. U.S.Patent Application Publication 20080066206). In contrast to siRNAs, aphased small RNA transcript is cleaved in phase. In contrast to miRNAs,a phased small RNA transcript is cleaved by DCL4 or a DCL4-likeorthologous ribonuclease (not DCL1) to produce multiple abundant smallRNAs capable of silencing a target gene. In contrast to the ta-siRNApathway, the phased small RNA locus transcribes to an RNA transcriptthat forms hybridized RNA independently of an RNA-dependent RNApolymerase and without a miRNA target site that initiates production ofdouble-stranded RNA. Novel recombinant DNA constructs that are designedbased on a phased small RNA locus are useful for suppression of one ormultiple target genes, without the use of miRNAs, ta-siRNAs, orexpression vectors designed to form a hairpin structure for processingto siRNAs. Furthermore, the recognition sites corresponding to a phasedsmall RNA are useful for suppression of a target sequence in a cell ortissue where the appropriate phased small RNA is expressed endogenouslyor as a transgene.

A. Virus Targets

In accordance with the invention, methods and compositions are providedfor conferring resistance to multiple viruses to plants, includingSolanaceous plants such as tomatoes. Viruses to which resistance may betargeted in the present invention include, without limitation, two ormore viruses from among the geminiviruses, tospovisues, andpotexviruses. FIG. 1 illustrates the genome organization ofrepresentatives of these viral genera.

1. Begomoviruses

The Geminiviridae are a large, diverse family of plant viruses thatinfect a broad variety of plants and cause significant crop lossesworldwide. They are characterized by twin icosahedral capsids andcircular ssDNA genomes that replicate through dsDNA intermediates.Geminiviruses (“begomovirus” and “geminivirus” are used interchangeablyherein) depend on the plant nuclear DNA and RNA polymerases forreplication and transcription. These viruses contribute only a fewfactors for their replication and transcription. The familyGeminiviridae contains three main genera (formerly termed “subgroups”)that differ with respect to insect vector, host range, and genomestructure.

Geminiviridae Subgroup I (genus Mastrevirus) includesleafhopper-transmitted viruses that generally infect monocot plants andhave single-component genomes.

Geminiviridae Subgroup III (genus Begomovirus) includeswhitefly-transmitted viruses that infect dicot plants and most commonlyhave bipartite genomes.

Geminiviridae Subgroup II (genus Curtovirus) viruses are transmitted byleafhoppers and have single-component genomes like Subgroup I, butinfect dicot plants like subgroup III.

2. Tospoviruses

Viruses in the genus Tospovirus cause significant worldwide crop losses.The genus name is derived from Tomato spotted wilt virus (“TSWV”). TheSpotted Wilt Disease of tomato was first observed in Australia in 1915and was later shown to be of viral origin. Until the early 1990s TSWVwas considered to be the sole member of the tomato spotted wilt group ofplant viruses. The identification and characterization of severalsimilar viruses, including Impatiens necrotic spot virus (INSV),Capsicum chlorosis virus (“CaCV”), Peanut bud necrosis virus (also knownas Groundnut bud necrosis virus, “GBNV”), and Tomato chlorotic spotvirus led to the creation of the plant-infecting tospovirus genus withinthe Bunyaviridae family. This family includes a large group ofpredominantly animal-infecting viruses. More than twenty tospoviruseshave since been identified and characterized and previously unknownspecies of the genus continue to be described on a regular basis.

Tospoviruses have a tripartite RNA genome of ambisense polarity. Thethree portions of the genome are termed the “L” segment, the “M”segment, and the “S” segment. A consensus terminal sequence of eachportion of the RNA genome is found, defined by segments UCUCGUUAGC (SEQID NO:167) at the 3′ end and AGAGCAAUCG (SEQ ID NO:168) at the 5′ end.The largest RNA, the “L segment,” encodes replicase. The medium sizeRNA, “M segment,” encodes glycoproteins G1 and G2 in thecomplementary-sense RNA and a nonstructural protein, NSm, in thegenome-sense RNA. The smallest segment, “S segment,” encodes thenucleocapsid protein (N) in the complementary-sense RNA and acell-to-cell movement, NSs, in the genome-sense RNA. The virus istransmitted by thrips in the genera Frankliniella (five species) andThrips (three species). Mechanical transmission of the virus is alsopossible. TSWV can infect more than 925 plant species belonging to 70botanical families, whereas the other tospovirus species have muchnarrower host ranges.

3. Potexviruses

The Pepino mosaic virus (PepMV) is a representative potexvirus fromamong the Flexiviridae, and is highly contagious with a significantpotential to cause damage in protected tomato production. Significantcrop losses are possible if action is not taken to eliminate infection.The virus is readily spread via contaminated tools, human hands orclothing, and by direct plant-to-plant contact. It can also betransmitted by grafting or when taking cuttings from infected motherplants. The use of coat protein-mediated resistance may provide goodresistance. However, including inverted repeats to the CP may enhanceresistant line production.

B. Nucleic Acid Compositions and Constructs

The invention provides recombinant DNA constructs and methods for use inachieving resistance to multiple (i.e. more than one) viral species andstrains from among the begomoviruses, tospoviruses, and potexviruses intransgenic plants. In certain embodiments, resistance is conferred to 2,3, 4, 5, 6, 7, 8, or more viral species selected from at least two ofthe following groups: begomoviruses, tospoviruses, and potexviruses. Theresistance may be directed by production of siRNA or miRNA, and may alsobe complemented by protein based approaches such as resistance mediatedby expressed coat protein or replicase, mutated forms of replicases, andproduction of aptamers. Genetically based tolerance (i.e. as identifiedin a classical breeding approach) may also be utilized.

As used herein, the term “nucleic acid” refers to a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesread from the 5′ to the 3′ end. The “nucleic acid” may also optionallycontain non-naturally occurring or altered nucleotide bases that permitcorrect read through by a polymerase and do not reduce expression of apolypeptide encoded by that nucleic acid. The term “nucleotide sequence”or “nucleic acid sequence” refers to both the sense and antisensestrands of a nucleic acid as either individual single strands or in theduplex. The term “ribonucleic acid” (RNA) is inclusive of dsRNA (doublestranded RNA), siRNA (small interfering RNA), shRNA (small hairpin RNA),mRNA (messenger RNA), miRNA (micro-RNA), tRNA (transfer RNA, whethercharged or discharged with a corresponding acylated amino acid), andcRNA (complementary RNA) and the term “deoxyribonucleic acid” (DNA) isinclusive of cDNA and genomic DNA and DNA-RNA hybrids. The words“nucleic acid segment,” “nucleotide sequence segment,” or more generally“segment” will be understood by those in the art as a functional termthat includes both genomic sequences, ribosomal RNA sequences, transferRNA sequences, messenger RNA sequences, operon sequences and smallerengineered nucleotide sequences that express or may be adapted toexpress, proteins, polypeptides or peptides.

As used herein, the term “substantially homologous” or “substantialhomology,” with reference to a nucleic acid sequence, includes anucleotide sequence that hybridizes under stringent conditions to any ofSEQ ID NOs:169-455, or a portion or complement thereof, are those thatallow an antiparallel alignment to take place between the two sequences,and the two sequences are then able, under stringent conditions, to formhydrogen bonds with corresponding bases on the opposite strand to form aduplex molecule that is sufficiently stable under conditions ofappropriate stringency, including high stringency, to be detectableusing methods well known in the art. Substantially homologous sequencesmay have from about 70% to about 80% sequence identity, or morepreferably from about 80% to about 85% sequence identity, or mostpreferable from about 90% to about 95% sequence identity, to about 99%sequence identity, to the referent nucleotide sequences as set forth thesequence listing, or the complements thereof.

As used herein, the term “ortholog” refers to a gene in two or morespecies that has evolved from a common ancestral nucleotide sequence,and may retain the same function in the two or more species.

As used herein, the term “sequence identity,” “sequence similarity” or“homology” is used to describe sequence relationships between two ormore nucleotide sequences. The percentage of “sequence identity” betweentwo sequences is determined by comparing two optimally aligned sequencesover a comparison window such as the full length of a referenced SEQ IDNO, wherein the portion of the sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison, and multiplyingthe result by 100 to yield the percentage of sequence identity. Asequence that is identical at every position in comparison to areference sequence is said to be identical to the reference sequence andvice-versa. A first nucleotide sequence when observed in the 5′ to 3′direction is said to be a “complement” of, or complementary to, a secondor reference nucleotide sequence observed in the 3′ to 5′ direction ifthe first nucleotide sequence exhibits complete complementarity with thesecond or reference sequence. As used herein, nucleic acid sequencemolecules are said to exhibit “complete complementarity” when everynucleotide of one of the sequences read 5′ to 3′ is complementary toevery nucleotide of the other sequence when read 3′ to 5′. A nucleotidesequence that is complementary to a reference nucleotide sequence willexhibit a sequence identical to the reverse complement sequence of thereference nucleotide sequence. These terms and descriptions are welldefined in the art and are easily understood by those of ordinary skillin the art.

As used herein, a “comparison window” refers to a conceptual segment ofat least 6 contiguous positions, usually about 50 to about 100, moreusually about 100 to about 150, in which a sequence is compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. The comparison window may compriseadditions or deletions (i.e. gaps) of about 20% or less as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences Those skilled in the artshould refer to the detailed methods used for sequence alignment, suchas in the Wisconsin Genetics Software Package Release 7.0 (GeneticsComputer Group, 575 Science Drive Madison, Wis., USA).

The present invention provides one or more DNA sequences capable ofbeing expressed as an RNA transcript in a cell or microorganism toinhibit target gene expression of at least one plant virus. Thesequences comprise a DNA molecule coding for one or more differentnucleotide sequences, wherein each of the different nucleotide sequencescomprises a sense nucleotide sequence and an antisense nucleotidesequence. The sequences may be connected by a spacer sequence. Thespacer sequence can constitute part of the sense nucleotide sequence orthe antisense nucleotide sequence or an unrelated nucleotide sequenceand forms within the dsRNA molecule between the sense and antisensesequences. The spacer sequence may comprise, for example, a sequence ofnucleotides of at least about 10-100 nucleotides in length, oralternatively at least about 100-200 nucleotides in length, at least200-400 about nucleotides in length, or at least about 400-500nucleotides in length. The sense nucleotide sequence or the antisensenucleotide sequence may be substantially identical to the nucleotidesequence of the target gene or a derivative thereof or a complementarysequence thereto. The dsDNA molecule may be placed operably under thecontrol of one or more promoter sequences that function in the cell,tissue or organ of the host expressing the dsDNA to produce RNAmolecules. As used herein, “expressing” or “expression” and the likerefer to transcription of a RNA molecule from a transcribedpolynucleotide. The RNA molecule may or may not be translated into apolypeptide sequence.

The invention also provides a DNA sequence for expression in a cell of aplant that, upon expression of the DNA to RNA and contact with a plantvirus achieves suppression of a target viral gene or viral replicationor symptomatology (i.e. expression of symptoms). Methods to express agene suppression molecule in plants are known (e.g. US Publication2006/0200878 A1; US Publication 2006/0174380; US Publication2008/0066206; Niu et al., 2006), and may be used to express a nucleotidesequence of the present invention.

Non-constitutive promoters suitable for use with recombinant DNAconstructs of the invention include spatially specific promoters,developmentally specific promoters, and inducible promoters. Spatiallyspecific promoters can include organelle-, cell-, tissue-, ororgan-specific promoters (e.g., a plastid-specific, a root-specific, apollen-specific, or a seed-specific promoter for suppressing expressionof the first target RNA in plastids, roots, pollen, or seeds,respectively). In many cases a seed-specific, embryo-specific,aleurone-specific, or endosperm-specific promoter is especially useful.Developmentally specific promoters can include promoters that tend topromote expression during certain developmental stages in a plant'sgrowth cycle, or at different seasons in a year. Inducible promotersinclude promoters induced by chemicals or by environmental conditionssuch as, but not limited to, biotic or abiotic stress (e.g., waterdeficit or drought, heat, cold, high or low nutrient or salt levels,high or low light levels, or pest or pathogen infection). Also ofinterest are microRNA promoters, especially those having adevelopmentally specific, spatially specific, or inducible expressionpattern. An expression-specific promoter can also include promoters thatare generally constitutively expressed but at differing degrees or“strengths” of expression, including promoters commonly regarded as“strong promoters” or as “weak promoters.”

Thus, a gene sequence or fragment for plant virus control according tothe invention may be cloned downstream of a promoter or promoters whichare operable in a transgenic plant cell and therein expressed to producemRNA in the transgenic plant cell that form dsRNA molecules. Numerousexamples of plant expressible promoters are known in the art (e.g. CaMV35S; FMV 35S; PC1SV (e.g. U.S. Pat. No. 5,850,019); ScBV; AtAct7, amongothers). Promoters useful for expression of polypeptides in plantsinclude those that are inducible, viral, synthetic, or constitutive asdescribed in Odell et al. (1985), and/or promoters that are temporallyregulated, spatially regulated, and spatio-temporally regulated. Anumber of organ-specific promoters have been identified and are known inthe art (e.g. U.S. Pat. Nos. 5,110,732; 5,837,848; 5,459,252; 6,229,067;Hirel et al. 1992). The dsRNA molecules contained in plant tissues areexpressed in a plant so that the intended suppression of the targetedvirus gene expression is achieved. The cauliflower mosaic virus 35Spromoter, an archetypal strong promoter common in transgenic plantapplications, or a related promoter such as the E35S or the FMVpromoter, may be employed for driving virus resistance genes. Promotershave also been identified that direct tissue specific gene expression.

A transgene transcription unit includes DNA sequence encoding a gene ofinterest. A gene of interest can include any coding or non-codingsequence from a virus species. Non-limiting examples of a non-codingsequence to be expressed by a transgene transcription unit include, butnot limited to, 5′ untranslated regions, promoters, enhancers, or othernon-coding transcriptional regions, 3′ untranslated regions,terminators, intron, microRNAs, microRNA precursor DNA sequences, smallinterfering RNAs, RNA components of ribosomes or ribozymes, smallnucleolar RNAs, RNA aptamers capable of binding to a ligand, and othernon-coding RNAs.

Non-limiting examples of a gene of interest further include, but are notlimited to, translatable (coding) sequence, such as genes encodingtranscription factors and genes encoding enzymes involved in thebiosynthesis or catabolism of molecules of interest (such as aminoacids, fatty acids and other lipids, sugars and other carbohydrates,biological polymers, and secondary metabolites including alkaloids,terpenoids, polyketides, non-ribosomal peptides, and secondarymetabolites of mixed biosynthetic origin). A gene of interest can be agene native to the cell (e.g., a plant cell) in which the recombinantDNA construct of the invention is to be transcribed, or can be anon-native gene. A gene of interest can be a marker gene, for example, aselectable marker gene encoding antibiotic, antifungal, or herbicideresistance, or a marker gene encoding an easily detectable trait (e.g.,in a plant cell, phytoene synthase or other genes imparting a particularpigment to the plant), or a gene encoding a detectable molecule, such asa fluorescent protein, luciferase, or a unique polypeptide or nucleicacid “tag” detectable by protein or nucleic acid detection methods,respectively). Selectable markers are genes of interest of particularutility in identifying successful processing of constructs of theinvention.

Genes of interest include those genes that may be described as “targetgenes.” The target gene can include a single gene or part of a singlegene that is targeted for suppression, or can include, for example,multiple consecutive segments of a target gene, multiple non-consecutivesegments of a target gene, multiple alleles of a target gene, ormultiple target genes from one or more species. The target gene can betranslatable (coding) sequence, or can be non-coding sequence (such asnon-coding regulatory sequence), or both. The transgene transcriptionunit can further include 5′ or 3′ sequence or both as required fortranscription of the transgene. In other embodiments (e.g., where it isdesirable to suppress a target gene across multiple strains or species,for instance of viruses), it may be desirable to design the recombinantDNA construct to be processed to a mature miRNA for suppressing a targetgene sequence common to the multiple strains or species in which thetarget gene is to be silenced. Thus, the miRNA processed from therecombinant DNA construct can be designed to be specific for one taxon(for example, specific to a genus, family, but not for other taxa.

The nucleic acid molecules or fragments of the nucleic acid molecules orother nucleic acid molecules in the sequence listing are capable ofspecifically hybridizing to other nucleic acid molecules under certaincircumstances. As used herein, two nucleic acid molecules are said to becapable of specifically hybridizing to one another if the two moleculesare capable of forming an anti-parallel, double-stranded nucleic acidstructure. A nucleic acid molecule is said to be the complement ofanother nucleic acid molecule if they exhibit complete complementarity.Two molecules are said to be “minimally complementary” if they canhybridize to one another with sufficient stability to permit them toremain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to becomplementary if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. Conventional stringencyconditions are described by Sambrook, et al. (1989), and by Haymes etal. (1985).

Departures from complete complementarity are therefore permissible for,as long as such departures do not completely preclude the capacity ofthe molecules to form a double-stranded structure. Thus, in order for anucleic acid molecule or a fragment of the nucleic acid molecule toserve as a primer or probe it needs only be sufficiently complementaryin sequence to be able to form a stable double-stranded structure underthe particular solvent and salt concentrations employed.

Appropriate stringency conditions that promote DNA hybridization are,for example, for applications requiring high selectivity, a relativelylow salt and/or high temperature condition, such as provided by about0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70°C. A high stringency condition, for example, is to wash thehybridization filter at least twice with high-stringency wash buffer(0.2×SSC or 1×SSC, 0.1% SDS, 65° C.). Other conditions, such as 6.0×sodium chloride/sodium citrate (SSC) at about 45° C., followed by a washof 2.0×SSC at 50° C., are also known to those skilled in the art or canbe found in Current Protocols in Molecular Biology (1989). For example,the salt concentration in the wash step can be selected from a lowstringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C. Both temperature andsalt may be varied, or either the temperature or the salt concentrationmay be held constant while the other variable is changed. A nucleic acidfor use in the present invention may specifically hybridize to one ormore of nucleic acid molecules from a plant virus selected from thegroup consisting of a tospovirus, a begomovirus, and a potexvirus, orcomplements thereof under such conditions. In specific embodiments, anucleic acid for use in the present invention will exhibit at least fromabout 80%, or at least from about 90%, or at least from about 95%, or atleast from about 98% or even about 100% sequence identity with one ormore nucleic acid molecules as set forth in the sequence listing, or acomplement thereof.

Nucleic acids of the present invention may also be synthesized, eithercompletely or in part, especially where it is desirable to provideplant-preferred sequences, by methods known in the art. Thus, all or aportion of the nucleic acids of the present invention may be synthesizedusing codons preferred by a selected host. Species-preferred codons maybe determined, for example, from the codons used most frequently in theproteins expressed in a particular host species. Other modifications ofthe nucleotide sequences may result in mutants having slightly alteredactivity.

DsRNA or siRNA nucleotide sequences comprise double strands ofpolymerized ribonucleotide and may include modifications to either thephosphate-sugar backbone or the nucleoside. Modifications in RNAstructure may be tailored to allow specific genetic inhibition. In oneembodiment, the dsRNA molecules may be modified through an enzymaticprocess so that siRNA molecules may be generated. Alternatively, aconstruct may be engineered to express a nucleotide segment for use inan miRNA- or siRNA-mediated resistance approach. The siRNA canefficiently mediate the down-regulation effect for some target genes insome pathogens. This enzymatic process may be accomplished by utilizingan RNAse III enzyme or a DICER enzyme, present in the cells of aninsect, a vertebrate animal, a fungus or a plant in the eukaryotic RNAipathway (Elbashir et al., 2001; Hamilton and Baulcombe, 1999). Thisprocess may also utilize a recombinant DICER or RNAse III introducedinto the cells of a target insect through recombinant DNA techniquesthat are readily known to the skilled in the art. Both the DICER enzymeand RNAse III, being naturally occurring in a pathogen or being madethrough recombinant DNA techniques, cleave larger dsRNA strands intosmaller oligonucleotides. The DICER enzymes specifically cut the dsRNAmolecules into siRNA pieces each of which is about 19-25 nucleotides inlength while the RNAse III enzymes normally cleave the dsRNA moleculesinto 12-15 base-pair siRNA. The siRNA molecules produced by the eitherof the enzymes have 2 to 3 nucleotide 3′ overhangs, and 5′ phosphate and3′ hydroxyl termini. The siRNA molecules generated by RNAse III enzymeare the same as those produced by Dicer enzymes in the eukaryotic RNAipathway and are hence then targeted and degraded by an inherent cellularRNA-degrading mechanism after they are subsequently unwound, separatedinto single-stranded RNA and hybridize with the RNA sequencestranscribed by the target gene. This process results in the effectivedegradation or removal of the RNA sequence encoded by the nucleotidesequence of the target gene in the pathogen. The outcome is thesilencing of a particularly targeted nucleotide sequence within thepathogen. Detailed descriptions of enzymatic processes can be found inHannon (2002).

A nucleotide sequence of the present invention can be recorded oncomputer readable media. As used herein, “computer readable media”refers to any tangible medium of expression that can be read andaccessed directly by a computer. Such media include, but are not limitedto: magnetic storage media, such as floppy discs, hard disc, storagemedium, and magnetic tape: optical storage media such as CD-ROM;electrical storage media such as RAM and ROM; optical characterrecognition formatted computer files, and hybrids of these categoriessuch as magnetic/optical storage media. A skilled artisan can readilyappreciate that any of the presently known computer readable mediums canbe used to create a manufacture comprising computer readable mediumhaving recorded thereon a nucleotide sequence of the present invention.

As used herein, “recorded” refers to a process for storing informationon computer readable medium. A skilled artisan can readily adopt any ofthe presently known methods for recording information on computerreadable medium to generate media comprising the nucleotide sequenceinformation of the present invention. A variety of data storagestructures are available to a skilled artisan for creating a computerreadable medium having recorded thereon a nucleotide sequence of thepresent invention. The choice of the data storage structure willgenerally be based on the means chosen to access the stored information.In addition, a variety of data processor programs and formats can beused to store the nucleotide sequence information of the presentinvention on computer readable medium. The sequence information can berepresented in a word processing text file, formatted incommercially-available software such as WordPerfect and Microsoft Word,or represented in the form of an ASCII text file, stored in a databaseapplication, such as DB2, Sybase, Oracle, or the like. The skilledartisan can readily adapt any number of data processor structuringformats (e.g. text file or database) in order to obtain computerreadable medium having recorded thereon the nucleotide sequenceinformation of the present invention.

Computer software is publicly available which allows a skilled artisanto access sequence information provided in a computer readable medium.Software that implements the BLAST (Altschul et al., 1990) and BLAZE(Brutlag, et al., 1993) search algorithms on a Sybase system can be usedto identify open reading frames (ORFs) within sequences such as theUnigenes and EST's that are provided herein and that contain homology toORFs or proteins from other organisms. Such ORFs are protein-encodingfragments within the sequences of the present invention and are usefulin producing commercially important proteins such as enzymes used inamino acid biosynthesis, metabolism, transcription, translation, RNAprocessing, nucleic acid and a protein degradation, proteinmodification, and DNA replication, restriction, modification,recombination, and repair.

As used herein, a “target,” a “target structural motif,” or a “targetmotif,” refers to any rationally selected sequence or combination ofsequences in which the sequences or sequence(s) are chosen based on athree-dimensional configuration that is formed upon the folding of thetarget motif or the nucleotide sequence thereof, as appropriate. Thereare a variety of target motifs known in the art.

C. Nucleic Acid Expression and Target Gene Suppression

The present invention provides, as an example, a transformed host plantof a pathogenic target organism, transformed plant cells and transformedplants and their progeny. The transformed plant cells and transformedplants may be engineered to express one or more of the dsRNA, miRNA, ormRNA sequences, under the control of a heterologous promoter, describedherein to provide a pathogen-protective effect. These sequences may beused for gene suppression in a pathogen, thereby reducing the level orincidence of disease caused by the pathogen on a protected transformedhost organism. As used herein the words “gene suppression” are intendedto refer to any of the well-known methods for reducing the levels ofprotein produced as a result of gene transcription to mRNA andsubsequent translation of the mRNA.

Gene suppression is also intended to mean the reduction of proteinexpression from a gene or a coding sequence includingposttranscriptional gene suppression and transcriptional suppression.Posttranscriptional gene suppression is mediated by the homology betweenof all or a part of a mRNA transcribed from a gene or coding sequencetargeted for suppression and the corresponding double stranded RNA usedfor suppression, and refers to the substantial and measurable reductionof the amount of available mRNA available in the cell for binding byribosomes or the prevention of translation by the ribosomes. Thetranscribed RNA can be in the sense orientation to effect what is calledco-suppression, in the anti-sense orientation to effect what is calledanti-sense suppression, or in both orientations producing a dsRNA toeffect what is called RNA interference (RNAi).

Gene suppression can also be effective against target genes in a plantvirus that may contact plant material containing gene suppressionagents, specifically designed to inhibit or suppress the expression ofone or more homologous or complementary sequences of the virus.Post-transcriptional gene suppression by anti-sense or sense orientedRNA to regulate gene expression in plant cells is disclosed in U.S. Pat.Nos. 5,107,065, 5,759,829, 5,283,184, and 5,231,020. The use of dsRNA tosuppress genes in plants is disclosed in WO 99/53050, WO 99/49029, U.S.Publication No. 2003/017596, U.S. Patent Application Publication2004/0029283.

A beneficial method of post transcriptional gene suppression versus aplant virus employs both sense-oriented and anti-sense-oriented,transcribed RNA which is stabilized, e.g., as a hairpin or stem and loopstructure (e.g. U.S. Publication 2007/0259785). A DNA construct foreffecting post transcriptional gene suppression may be one in which afirst segment encodes an RNA exhibiting an anti-sense orientationexhibiting substantial identity to a segment of a gene targeted forsuppression, which is linked to a second segment encoding an RNAexhibiting substantial complementarity to the first segment. Such aconstruct forms a stem and loop structure by hybridization of the firstsegment with the second segment and a loop structure from the nucleotidesequences linking the two segments (see WO94/01550, WO98/05770, US2002/0048814, and US 2003/0018993). Co-expression with an additionaltarget gene segment may also be employed, as noted above (e.g.WO05/019408).

According to one embodiment of the present invention, there is providedan exogenous nucleotide sequence (i.e. not naturally found in the genomeof the host plant cell), for which expression results in transcriptionof a RNA sequence that is substantially similar in sequence to a RNAmolecule of a targeted gene of a plant virus, selected from the groupconsisting of a tospovirus, a begomovirus, and a potexvirus, thatcomprises an RNA sequence encoded by a nucleotide sequence within thegenome of the virus. By substantially similar is meant that theexogenous RNA sequence is capable of effecting RNA-mediated genesuppression of a target sequence in a viral genome. Thus, adown-regulation of the expression of the nucleotide sequencecorresponding to the target gene is effected.

In certain embodiments of the invention, expression of a fragment of atleast 21 contiguous nucleotides of a nucleic acid sequence of any of SEQID NOs:169-455, or complements thereof, may be utilized, includingexpression of a fragment of up to 21, 36, 60, 100, 550, or 1000contiguous nucleotides, or sequences displaying 90-100% identity withsuch sequences, or their complements. In specific embodiments, anucleotide provided by the invention may comprise a sequence selectedfrom among SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55, 59, 63,67, 71, 85, 99, 113, 127, and 141. In other specific embodiments, anucleotide provided by the invention may comprise a sequence selectedfrom among SEQ ID NOs: 379-455. In yet other embodiments, a nucleotideprovided by the invention may be described as comprising one or more ofnucleotides 1-21, 22-50, 51-100, 101-150, 151-200, 201-250, 251-300,301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700,701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050,1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350,1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650,1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950,1951-2000, 2001-2050, 2051-2100, 23-75, 76-125, 126-175, 176-225,226-275, 276-325, 326-375, 376-425, 426-475, 476-525, 526-575, 576-625,626-675, 676-725, 726-775, 776-825, 826-875, 876-925, 926-975, 976-1025,1026-1075, 1076-1125, 1126-1175, 1176-1225, 1226-1275, 1276-1325,1326-1375, 1376-1425, 1426-1475, 1476-1525, 1526-1575, 1576-1625,1626-1675, 1676-1725, 1726-1775, 1776-1825, 1826-1875, 1876-1925,1926-1975, 1976-2025, 2026-2075, 2076-2125, 1-550, 200-750, 300-850,400-950, 500-1050, 600-1150, 700-1250, 800-1350, 900-1450, 1000-1550,1100-1650, 1200-1750, 1300-1850, 1400-1950, 1500-2050, up to the fulllength of the sequence, of one or more of any of SEQ ID NOs:169-455. Asequence complementary to all or a portion of any one or more of SEQ IDNOs:169-455, or SEQ ID NOs: 1, 7, 13, 19, 25, 31, 37, 43, 47, 51, 55,59, 63, 67, 71, 85, 99, 113, 127, and 141, wherein expression of saidsequence suppresses the expression of any one or more of gene(s) encodedby a nucleotide sequence of SEQ ID NOs:169-455, is contemplated. Thesequences arrayed for expression to produce dsRNA can be combined with:(1) sequences designed for production of miRNA, including in stackedmiRNA cassettes; and/or (2) sequences for inhibition of viral assembly,in order to synergistically inhibit target viruses. Methods forselecting specific sub-sequences as targets for miRNA or siRNA-mediatedgene suppression are known in the art (e.g. Reynolds et al., 2004).

Inhibition of a target gene using dsRNA technology of the presentinvention is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted forsuppression. RNA containing a nucleotide sequences identical to aportion of the target gene transcript is usually preferred forinhibition. RNA sequences with insertions, deletions, and single pointmutations relative to the target sequence have also been found to beeffective for inhibition. In performance of the present invention, theinhibitory dsRNA and the portion of the target gene may share at leastfrom about 80% sequence identity, or from about 90% sequence identity,or from about 95% sequence identity, or from about 99% sequenceidentity, or even about 100% sequence identity. Alternatively, theduplex region of the RNA may be defined functionally as a nucleotidesequence that is capable of hybridizing with a portion of the targetgene transcript. A less than full length sequence exhibiting a greaterhomology compensates for a longer less homologous sequence. The lengthof the identical nucleotide sequences may be at least about 18, 21, 25,50, 100, 200, 300, 400, 500 or at least about 1000 bases. Normally, asequence of greater than 20-100 nucleotides should be used, though asequence of greater than about 200-300 nucleotides may be preferred, anda sequence of greater than about 500-1000 nucleotides may be especiallypreferred depending on the size of the target gene. The invention hasthe advantage of being able to tolerate sequence variations that mightbe expected due to genetic mutation, strain polymorphism, orevolutionary divergence. The introduced nucleic acid molecule may notneed to be absolutely homologous to the target sequence, and it may notneed to be full length relative to either the primary transcriptionproduct or fully processed mRNA of the target gene. Therefore, thoseskilled in the art need to realize that, as disclosed herein, 100%sequence identity between the RNA and the target gene is not required topractice the present invention.

Inhibition of target gene expression may be quantified by measuringeither the endogenous target RNA or the protein produced by translationof the target RNA and the consequences of inhibition can be confirmed byexamination of the outward properties of the cell or organism.Techniques for quantifying RNA and proteins are well known to one ofordinary skill in the art.

In certain embodiments gene expression is inhibited by at least 10%, byat least 33%, by at least 50%, or by at least 80%. In particularembodiments of the invention gene expression is inhibited by at least80%, by at least 90%, by at least 95%, or by at least 99% within hostcells infected by the virus, such a significant inhibition takes place.Significant inhibition is intended to refer to sufficient inhibitionthat results in a detectable phenotype (e.g., reduction of symptomexpression, etc.) or a detectable decrease in RNA and/or proteincorresponding to the target gene being inhibited.

DsRNA molecules may be synthesized either in vivo or in vitro. The dsRNAmay be formed by a single self-complementary RNA strand or from twocomplementary RNA strands. Endogenous RNA polymerase of the cell maymediate transcription in vivo, or cloned RNA polymerase can be used fortranscription in vivo or in vitro. Inhibition may be targeted byspecific transcription in an organ, tissue, or cell type; stimulation ofan environmental condition (e.g., infection, stress, temperature,chemical inducers); and/or engineering transcription at a developmentalstage or age. The RNA strands may or may not be polyadenylated; the RNAstrands may or may not be capable of being translated into a polypeptideby a cell's translational apparatus.

As used herein, the term “disease control agent,” or “gene suppressionagent” refers, in certain embodiments, to a particular RNA moleculeconsisting of a first RNA segment and a second RNA segment linked by athird RNA segment. The first and the second RNA segments lie within thelength of the RNA molecule and are substantially inverted repeats ofeach other and are linked together by the third RNA segment. Thecomplementarity between the first and the second RNA segments results inthe ability of the two segments to hybridize in vivo and in vitro toform a double stranded molecule, i.e., a stem, linked together at oneend of each of the first and second segments by the third segment whichforms a loop, so that the entire structure forms into a stem and loopstructure, or even more tightly hybridizing structures may form into astem-loop knotted structure. The first and the second segmentscorrespond invariably, but not necessarily respectively, to a sense andan antisense sequence homologous with respect to the target RNAtranscribed from the target gene in the target virus that is intended tobe suppressed by the dsRNA molecule.

As used herein, the term “genome” as it applies to a plant virus or ahost encompasses not only viral DNA or RNA, or chromosomal DNA foundwithin the nucleus, but organelle DNA found within subcellularcomponents of the cell. The DNA's of the present invention introducedinto plant cells can therefore be either chromosomally integrated ororganelle-localized. The term “genome” as it applies to bacteriaencompasses both the chromosome and plasmids within a bacterial hostcell. The DNA's of the present invention introduced into bacterial hostcells can therefore be either chromosomally integrated orplasmid-localized.

It is envisioned that the compositions of the present invention can beincorporated within the seeds of a plant species either as a product ofexpression from a recombinant gene incorporated into a genome of theplant cells. The plant cell containing a recombinant gene is consideredherein to be a transgenic event.

D. Recombinant Vectors and Host Cell Transformation

A recombinant DNA vector may, for example, be a linear or a closedcircular plasmid. The vector system may be a single vector or plasmid ortwo or more vectors or plasmids that together contain the total DNA tobe introduced into the genome of the bacterial host. In addition, abacterial vector may be an expression vector. Nucleic acid molecules asset forth in the sequence listing, or complements or fragments thereof,can, for example, be suitably inserted into a vector under the controlof a suitable promoter that functions in one or more microbial hosts todrive expression of a linked coding sequence or other DNA sequence. Manyvectors are available for this purpose, and selection of the appropriatevector will depend mainly on the size of the nucleic acid to be insertedinto the vector and the particular host cell to be transformed with thevector. Each vector contains various components depending on itsfunction (amplification of DNA or expression of DNA) and the particularhost cell with which it is compatible. The vector components forbacterial transformation generally include, but are not limited to, oneor more of the following: a signal sequence, an origin of replication,one or more selectable marker genes, and an inducible promoter allowingthe expression of exogenous DNA.

Expression and cloning vectors generally contain a selection gene, alsoreferred to as a selectable marker. This gene encodes a proteinnecessary for the survival or growth of transformed host cells grown ina selective culture medium. Typical selection genes encode proteins that(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.Those cells that are successfully transformed with a heterologousprotein or fragment thereof produce a protein conferring drug resistanceand thus survive the selection regimen.

An expression vector for producing a mRNA can also contain an induciblepromoter that is recognized by a host organism and is operably linked tothe nucleic acid. The term “operably linked,” as used in reference to aregulatory sequence and a structural nucleotide sequence, means that theregulatory sequence causes regulated expression of the linked structuralnucleotide sequence. “Regulatory sequences” or “control elements” referto nucleotide sequences located upstream (5′ noncoding sequences),within, or downstream (3′ non-translated sequences) of a structuralnucleotide sequence, and which influence the timing and level or amountof transcription, RNA processing or stability, or translation of theassociated structural nucleotide sequence. Regulatory sequences mayinclude promoters, translation leader sequences, introns, enhancers,stem-loop structures, repressor binding sequences, and polyadenylationrecognition sequences and the like.

Construction of suitable vectors containing one or more of theabove-listed components employs standard recombinant DNA techniques.Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligatedin the form desired to generate the plasmids required. Examples ofavailable bacterial expression vectors include, but are not limited to,the multifunctional E. coli cloning and expression vectors such asBluescript™ (Stratagene, La Jolla, Calif.), in which, for example, anucleic acid, or fragment thereof may be ligated into the vector inframe with sequences for the amino-terminal Met and the subsequent 7residues of β-galactosidase so that a hybrid protein is produced; pINvectors (Van Heeke and Schuster, 1989); and the like.

The present invention also contemplates transformation of a nucleotidesequence of the present invention into a plant to achievevirus-inhibitory levels of expression of one or more RNA molecules. Atransformation vector can be readily prepared using methods available inthe art. The transformation vector comprises one or more nucleotidesequences that is/are capable of being transcribed to an RNA moleculeand that is/are substantially homologous and/or complementary to one ormore nucleotide sequences encoded by the genome of the target virus orviruses, such that upon contact of the RNA transcribed from the one ormore nucleotide sequences by the target plant-parasitic virus, there isdown-regulation of expression of at least one of the respectivenucleotide sequences of the genome of the virus.

The transformation vector may be termed a dsDNA construct and may alsobe defined as a recombinant molecule, a disease control agent, a geneticmolecule or a chimeric genetic construct. A chimeric genetic constructof the present invention may comprise, for example, nucleotide sequencesencoding one or more antisense transcripts, one or more sensetranscripts, one or more of each of the aforementioned, wherein all orpart of a transcript there from is homologous to all or part of an RNAmolecule comprising an RNA sequence encoded by a nucleotide sequencewithin the genome of a plant virus.

In one embodiment a plant transformation vector comprises an isolatedand purified DNA molecule comprising a heterologous promoter operativelylinked to one or more nucleotide sequences of the present invention. Thenucleotide sequence includes a segment coding all or part of an RNApresent within a targeted RNA transcript and may comprise invertedrepeats of all or a part of a targeted RNA. The DNA molecule comprisingthe expression vector may also contain a functional intron sequencepositioned either upstream of the coding sequence or even within thecoding sequence, and may also contain a five prime (5′) untranslatedleader sequence (i.e., a UTR or 5′-UTR) positioned between the promoterand the point of translation initiation.

A plant transformation vector may contain sequences from more than onegene, thus allowing production of more than one dsRNA, miRNA, or siRNAfor inhibiting expression of genes of the more than one target virus.For instance a vector or construct may comprise up to about 8 or 10, ormore, nucleic acid segments for transcription of antiviral sequences, asshown in FIG. 10. One skilled in the art will readily appreciate thatsegments of DNA whose sequence corresponds to that present in differentgenes can be combined into a single composite DNA segment for expressionin a transgenic plant. Alternatively, a plasmid of the present inventionalready containing at least one DNA segment can be modified by thesequential insertion of additional DNA segments between the enhancer andpromoter and terminator sequences. In the disease control agent of thepresent invention designed for the inhibition of multiple genes, thegenes to be inhibited can be obtained from the same plant viral strainor species in order to enhance the effectiveness of the control agent.In certain embodiments, the genes can be derived from different plantviruses in order to broaden the range of viruses against which theagent(s) is/are effective. When multiple genes are targeted forsuppression or a combination of expression and suppression, apolycistronic DNA element can be fabricated as illustrated and disclosedin U.S. Publication No. 2004/0029283.

A recombinant DNA vector or construct of the present invention maycomprise a selectable marker that confers a selectable phenotype onplant cells. Selectable markers may also be used to select for plants orplant cells that contain the exogenous nucleic acids encodingpolypeptides or proteins of the present invention. The marker may encodebiocide resistance, antibiotic resistance (e.g., kanamycin, G418bleomycin, hygromycin, etc.), or herbicide resistance (e.g., glyphosate,etc.). Examples of selectable markers include, but are not limited to, aneo gene which codes for kanamycin resistance and can be selected forusing kanamycin, G418, etc., a bar gene which codes for bialaphosresistance; a mutant EPSP synthase gene which encodes glyphosateresistance; a nitrilase gene which confers resistance to bromoxynil; amutant acetolactate synthase gene (ALS) which confers imidazolinone orsulfonylurea resistance; and a methotrexate resistant DHFR gene.Multiple selectable markers are available that confer resistance toampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin,kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin,spectinomycin, rifampicin, and tetracycline, and the like. Examples ofsuch selectable markers are illustrated in U.S. Pat. Nos. 5,550,318;5,633,435; 5,780,708 and 6,118,047.

A recombinant vector or construct of the present invention may alsoinclude a screenable marker. Screenable markers may be used to monitorexpression. Exemplary screenable markers include a β-glucuronidase oruidA gene (GUS) which encodes an enzyme for which various chromogenicsubstrates are known (Jefferson et al., 1987); one or more of thevarious fluorescent proteins (FP) genes such as green fluorescentprotein (GFP), red fluorescent protein (RFP) or any one of a largefamily of proteins which typically fluoresce at a characteristicwavelength; an R-locus gene, which encodes a product that regulates theproduction of anthocyanin pigments (red color) in plant tissues(Dellaporta et al., 1988); a β-lactamase gene (Sutcliffe et al., 1978),a gene which encodes an enzyme for which various chromogenic substratesare known (e.g., PADAC, a chromogenic cephalosporin); a luciferase gene(Ow et al., 1986) a xylE gene (Zukowski et al., 1983) which encodes acatechol dioxygenase that can convert chromogenic catechols; anα-amylase gene (Ikatu et al., 1990); a tyrosinase gene (Katz et al.,1983) which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone which in turn condenses to melanin; an α-galactosidase,which catalyzes a chromogenic α-galactose substrate.

Plant transformation vectors for use with the present invention may forinstance include those derived from a Ti plasmid of Agrobacteriumtumefaciens (e.g. U.S. Pat. Nos. 4,536,475, 4,693,977, 4,886,937,5,501,967 and EP 0 122 791). Agrobacterium rhizogenes plasmids (or “Ri”)are also useful and known in the art. Other preferred planttransformation vectors include those disclosed, e.g., byHerrera-Estrella (1983); Bevan (1983), Klee (1985) and EP 0 120 516.

In certain embodiments, a functional recombinant DNA may be introducedat a non-specific location in a plant genome. In special cases it may beuseful to insert a recombinant DNA construct by site-specificintegration. Several site-specific recombination systems exist which areknown to function in plants include cre-lox as disclosed in U.S. Pat.No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695.

Suitable methods for transformation of host cells for use with thecurrent invention are believed to include virtually any method by whichDNA can be introduced into a cell (see, for example, Miki et al., 1993),such as by transformation of protoplasts (U.S. Pat. No. 5,508,184;Omirulleh et al., 1993), by desiccation/inhibition-mediated DNA uptake(Potrykus et al., 1985), by electroporation (U.S. Pat. No. 5,384,253),by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S.Pat. No. 5,302,523; and U.S. Pat. No. 5,464,765), byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055;5,591,616; 5,693,512; 5,824,877; 5,981,840; 6,384,301) and byacceleration of DNA coated particles (U.S. Pat. Nos. 5,015,580;5,550,318; 5,538,880; 6,160,208; 6,399,861; 6,403,865; Padgette et al.1995), etc. Through the application of techniques such as these, thecells of virtually any species may be stably transformed. In the case ofmulticellular species, the transgenic cells may be regenerated intotransgenic organisms.

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium (for example, Horsch et al., 1985). A. tumefaciens and A.rhizogenes are plant pathogenic soil bacteria which geneticallytransform plant cells. The Ti and Ri plasmids of A. tumefaciens and A.rhizogenes, respectively, carry genes responsible for genetictransformation of the plant. Descriptions of Agrobacterium vectorsystems and methods for Agrobacterium-mediated gene transfer areprovided by numerous references, including Gruber et al. 1993; Miki etal., 1993, Moloney et al., 1989, and U.S. Pat. Nos. 4,940,838 and5,464,763. Other bacteria such as Sinorhizobium, Rhizobium, andMesorhizobium that interact with plants naturally can be modified tomediate gene transfer to a number of diverse plants. Theseplant-associated symbiotic bacteria can be made competent for genetransfer by acquisition of both a disarmed Ti plasmid and a suitablebinary vector (Broothaerts et al., 2005). Methods for introducing virussequences to plants may also be used (e.g. Grimsley, 1990, Boulton,1996).

Methods for the creation of transgenic plants and expression ofheterologous nucleic acids in plants in particular are known and may beused with the nucleic acids provided herein to prepare transgenic plantsthat exhibit reduced susceptibility to plant-pathogenic viruses. Planttransformation vectors can be prepared, for example, by inserting thedsRNA producing nucleic acids disclosed herein into plant transformationvectors and introducing these into plants. One known vector system hasbeen derived by modifying the natural gene transfer system ofAgrobacterium tumefaciens. The natural system comprises large Ti(tumor-inducing)-plasmids containing a large segment, known as T-DNA,which is transferred to transformed plants. Another segment of the Tiplasmid, the vir region, is responsible for T-DNA transfer. The T-DNAregion is bordered by terminal repeats. In the modified binary vectorsthe tumor-inducing genes have been deleted and the functions of the virregion are utilized to transfer foreign DNA bordered by the T-DNA bordersequences. The T-region may also contain a selectable marker forefficient recovery of transgenic plants and cells, and a multiplecloning site for inserting sequences for transfer such as a dsRNAencoding nucleic acid.

Protocols for transformation of tomato cells are known in the art (e.g.McCormick, 1991). Alternate plant transformation protocols are discussedin Boulton (1996), and Grimsley (1990). Transformation and regenerationprotocols for other plants, such as pepper, are known in the art (e.g.Christopher and Rajam, 1996; U.S. Pat. No. 5,262,316; Liu et al. 1990).For instance, such a protocol for transformation of tomato could includewell known steps of seed sterilization, seed germination and growth,explanting of seedlings, Agrobacterium culture growth and preparation,co-cultivation, selection, and regeneration.

E. Transgenic Plants and Cells

A transgenic plant formed using Agrobacterium transformation methodstypically may contain a single simple recombinant DNA sequence insertedinto one chromosome, referred to as a transgenic event. Such transgenicplants can be referred to as being heterozygous for the insertedexogenous sequence. A transgenic plant homozygous with respect to atransgene can be obtained by sexually mating (selfing) an independentsegregant transgenic plant that contains a single exogenous genesequence to itself, for example an F0 plant, to produce F1 seed. Onefourth of the F1 seed produced will be homozygous with respect to thetransgene. Germinating F1 seed results in plants that can be tested forheterozygosity, typically using a SNP assay or a thermal amplificationassay that allows for the distinction between heterozygotes andhomozygotes (i.e., a zygosity assay). Crossing a heterozygous plant withitself or another heterozygous plant results in heterozygous progeny, aswell as homozygous transgenic and homozygous null progeny.

In addition to direct transformation of a plant with a recombinant DNAconstruct, transgenic plants can be prepared by crossing a first planthaving a recombinant DNA construct with a second plant lacking theconstruct. For example, recombinant DNA for gene suppression can beintroduced into first plant line that is amenable to transformation toproduce a transgenic plant that can be crossed with a second plant lineto introgress the recombinant DNA for gene suppression into the secondplant line.

F. Virus Resistance Screens

Inoculation and disease testing with viruses such as TSWV, TYLCV, andPepMV was performed using either mechanical transmission oragroinfection (e.g. Boulton, 1996; Grimsley, 1990). TSWV infection wasaccomplished mechanically, essentially as described by Kumar et al.,(1993) with some modifications. Inoculation with begomovirus (e.g.TYLCV) and disease testing of plants was accomplished by agroinfectionas follows: Seeds of tomato (Lycopersicon esculentum) cvs. (Resistant(R): HP919; Intermediate Resistant (IR): Hilario; Susceptible (S):Arletta) were sown and approximately 20 plants for each inoculation, aswell as a non-inoculated or mock-inoculated control, were grown for 7-10days, to the cotyledon stage with no primary leaf visible. The lowersides of the cotyledons were then infiltrated using a needlelesssyringe, and additional inoculations were subsequently performed byinjection into stems at the 2-3 true leaf stage, approximately 1-2 weekslater. Infiltration or injection utilized a transformed A. tumefaciensstrain, induced with acetosyringone, containing an infections clone ofthe virus that had been grown in YEB broth with antibiotics forselection of the pBIN vector for about 15 hours at 28° C. on a shaker(170 RPM) from inoculation of a fresh culture. Following growth at 28°C., the culture was spun down and resuspended in 10 ml MMA (Per 1 liter:20 g sucrose, 5 g MS salts, 1.95 g MES, pH 5.6 with NaOH, and 1 ml of200 mM acetosyringone stock; dH₂O to 1 liter). Plants were grown at20-25° C. (day/night), 16 hours light in the greenhouse or growthchamber. After 6 weeks, symptoms were scored as follows:

1 no symptoms visible (resistant)

3. very mild symptoms (resistant)

5. medium symptoms

7. strong symptoms

9. very strong symptoms

Inoculation with the potexvirus Pepino mosaic virus (PepMV) wasaccomplished as follows: Virus inoculum was prepared by mechanicallyinfecting tomato seedlings (susceptible cv. Apollo, 9-12 days old)following dusting of leaves with carborundum powder. Infected tissueswere harvested and 1 g of infected tissue was homogenized with 5-10 mlof phosphate buffer (pH 9). This prepared inoculum was then used tomechanically inoculate experimental plants at the cotyledon stage (7-10days after sowing). Inoculated plants were grown in the greenhouse at19-23° C. (day/night), with 16 hours of light per 24 hours, ingreenhouse with 70% relative humidity. Plants were evaluated at 11-21days after inoculation, and scored as follows:

1 no symptoms visible (resistant)

3. some foliar chlorosis

5. chlorosis in foliar veins and/or foliar mosaic

7. foliar vein chlorosis and spiky leaves

9. foliar vein chlorosis and spiky leaves and yellow mosaic

G. RNA Extraction and siRNA Northern Blots

RNA was extracted for small RNA northern blotting using Trizol®essentially according to the manufacturer's directions (Invitrogen).Briefly, approximately 100-200 mg of fresh leaf tissue was ground inliquid nitrogen in 1.5 ml centrifuge tube placed on dry ice. Sample wasremoved from dry ice and 1 ml of Trizol was added under a fume hood.This was mixed well and incubated at room temperature (RT) for 10minutes. Next, 0.2 ml of chloroform was added, and the sample was shakenby hand for 30 seconds and centrifuged at 13000 rpm in a refrigeratedtable top centrifuge for 15 minutes at 4° C.

The aqueous phase was transferred to a new tube, 0.5 ml of isopropanolwas added, and tubes were inverted a few times, and then incubated for10 min at RT followed by centrifugation at 13000 rpm in a refrigeratedtable top centrifuge for 15 min at 4° C. The supernatant was discardedand the pellet was washed by adding 0.75 ml of 75% ethanol, thencentrifuged at 13000 rpm for 10 min at 4° C. The supernatant wasdiscarded and RNA was air dried for 10 minutes at room temperature.

The pellet was resuspended by adding 100 μl of RNAse free ddH2O and wasvortexed for a few seconds. The samples were frozen on dry ice and theRNA concentrated by the use of a speed vacuum for about 20 minutes: thisstep removes all traces of ethanol. RNA was quantified byspectrophotometer (usually about 80-100 μg are recovered from tomatoleaf). About 7 μg of total RNA were loaded for siRNA analysis.

siRNA Northern blot with DIG labeled probe: Pre-run the 15% TBE-Urea gel(Invitrogen EC68855BOX) for about 30′ at 110 volts. Samples wereprepared for loading by adding 7 μl of Novex® TBE-Urea Sample Buffer 2×(Invitrogen LC6876) to 7 μl of total RNA (5-7 μg), they are denaturedfor about 10′ at 94° C. and immediately cooled on ice. If samplevisualization under UV light was needed, ethidium bromide was added tothe sample buffer (1 μl of EtBr @0.624 mg/ml for every 100 μl ofbuffer). Samples were briefly spun down before loading into the gelwells and electrophoresis was carried on for about 1.5 hrs at 180 Voltsin 0.5×TBE until the blue dye reached the bottom of the gel. After therun was terminated, the gel was observed under UV lights.

Transfer of the gel to Nytran Supercharge Nylon membrane (VWR 28151-318)was done in a Transblot semi dry transfer cell (BioRad 170-3940): themembrane was pre-wet in water and equilibrated in 0.5×TBE together with2 pieces of extra-thick paper (BioRad 170-3968). The transfer was set upaccording to manufacturer's instruction (anode-blottingpaper-membrane-gel-blotting paper-cathode) and carried on for 50′ at 380mAmp. After transfer the membrane was left to air dry for about 10′ andthen the RNA was cross-linked to it in a Stratalinker 1800 (Stratagene).

Hybridization: The membrane was pre-hybridized at 42° C. for 1 hr with10 ml of PerfectHyb solution (Sigma H7033) in a hybridization oven. 200ng of DIG labeled probe, prepared with the PCR DIG labeling mix (Roche11585550910) following manufacturer's instruction, was denatured for 10′at 94° C., cooled on ice, and added to 10 ml of fresh hybridizationsolution, which was added to the pre-hybridized membrane and incubatedovernight at 42° C. in the hybridization oven.

Washes and detection: After discarding the hybridization solution, themembranes was briefly rinsed in 2×SSC 0.1% SDS; and then washed 2 timesin pre-warmed 2×SSC 0.1% SDS for 20′ at 50° C., 2 times in pre-warmed1×SSC 0.1% SDS for 20′ at 50° C., 2 times in pre-warmed 0.5×SSC 0.1% SDSfor 20′ at 50° C. Detection was performed following the instructionsprovided with the DIG Wash and Block Buffer Set (Roche 11585762001).Briefly, the membrane was rinsed for 2′ in 1×DIG Washing Buffer beforeincubating for 1 hr at RT in 100 mL DIG Blocking Solution (10 mL 10×Blocking buffer+10 mL 10× Maleic Acid+80 mL water). It was thenincubated for 1 hr at room temperature in 100 mL fresh DIG BlockingSolution to which 10 μl of Anti-Digoxigenin-AP antibody (Roche11093274910) had been added. The membrane was washed 15′ at RT twice 100mL 1×DIG Washing Buffer and equilibrated with 100 mL of 1×DIG DetectionBuffer. About 1 mL of CDP-star solution (Roche 12041677001) wasdistributed across the top of the membrane, which was placed between 2plastic sheets, incubated at RT for 5′ and exposed to a film forvariable amounts of time before developing.

H. Validation of Constructs in Transgenic Tomato Plants

The engineered sequences for generating dsRNA or miRNA were cloned intobinary vectors for tomato transformation. R₀ transgenic plants wereassayed for the production of the specific ˜21mers and/or virusresistance efficacy by small RNA northern blot analysis and resistanceassays as outlined above and as described in Example 7. Other selecteddsRNA-generating sequences or miRNA-generating sequences, for instancefrom among any of SEQ ID NOs:1-154, or from within SEQ ID NOs:169-455 orportions thereof, may be utilized to create additional efficaciousconstructs. Thus, for instance, MIR backbone sequence(s), for instanceMON1 from soybean, MONS from soybean, MON13 from rice, MON18 from maize,miR159a from maize, or mi167 g from maize (SEQ ID NOs:155, 157, 159,161, 163, or 165) may be utilized to create dsRNA- or miRNA-generatingsequence(s). In certain embodiments, the dsRNA-generating sequence ormiRNA-generating seqence may comprise any of SEQ ID NOs:1, 7, 13, 19,25, 31, 37, 43, 47, 55, 59, 63, 67, 71, 85, 99, 113, 127, 141, or379-455. In particular embodiments, the sequence may comprise any of SEQID NOs:156, 158, 160, 162, 164, 166, 363, 364, 365, 366, 367, 368, 369,370, 371, 372, 373, 374, or 375.

The present invention includes combinations with other disease controltraits in a plant, including non-transgenic approaches, to achievedesired traits for enhanced control of plant disease. Combining diseasecontrol traits that employ distinct modes-of-action can provideprotected transgenic plants with superior durability over plantsharboring a single control trait because of the reduced probability thatresistance will develop in the field. Thus, in certain embodiments, atleast two or at least three modes of action are employed to confer virusresistance, wherein such modes of action are selected from the groupconsisting of expression of dsRNA, expression of miRNA, inhibition ofvirion assembly, phenotypic expression of a non-transgenic virusresistance trait, and transgenic protein expression. In particularembodiments, transgenic protein expression comprises expression of acoat protein-encoding nucleic acid sequence, expression of a movementprotein-encoding sequence or expression of a replicase-encoding nucleicacid sequence.

The invention also relates to commodity products containing one or moreof the sequences of the present invention, and produced from arecombinant plant or seed containing one or more of the nucleotidesequences of the present invention are specifically contemplated asembodiments of the present invention. A commodity product containing oneor more of the sequences of the present invention is intended toinclude, but not be limited to, meals, oils, crushed or whole grains orseeds of a plant, or any food product comprising any meal, oil, orcrushed or whole grain of a recombinant plant or seed containing one ormore of the sequences of the present invention. The detection of one ormore of the sequences of the present invention in one or more commodityor commodity products contemplated herein is defacto evidence that thecommodity or commodity product is composed of a transgenic plantdesigned to express one or more of the nucleotides sequences of thepresent invention for the purpose of controlling plant disease usingdsRNA mediated gene suppression methods.

I. Nucleic Acid Compositions

The present invention provides methods for obtaining a nucleic acidcomprising a nucleotide sequence for producing a dsRNA, miRNA, or siRNA.In one embodiment, such a method comprises: (a) analyzing one or moretarget gene(s) for their expression, function, and phenotype upondsRNA-mediated, or mi-RNA- or siRNA-mediated suppression of a gene of aplant pathogenic virus; (b) probing a nucleic acid library with ahybridization probe comprising all or a portion of a nucleotidesequence, or a homolog thereof, from a targeted virus that displays analtered phenotype in a dsRNA-mediated suppression analysis; (c)identifying a DNA clone that hybridizes with the hybridization probe;(d) isolating the DNA clone identified in step (b); and (e) sequencingthe nucleic acid fragment that comprises the clone isolated in step (d)wherein the sequenced nucleic acid molecule transcribes all or asubstantial portion of the RNA nucleotide acid sequence or a homologthereof. The RNA-mediated resistance approach utilizing dsRNA, miRNA, orsiRNA may be supplemented by placing antiviral sequences into the loopof a dsRNA-encoding sequence, or by inserting a sequence that encodes anefficacious dsRNA or miRNA into an intron of a polypeptide expressioncassette (intronic dsRNA/miRNA; Frizzi et al., 2008) (e.g. FIG. 9). Forexample, viral protein(s) such as coat protein and/or replicase may beexpressed in a transgenic plant that also expresses an efficaciousdsRNA, miRNA, or siRNA, without use of an additional transgene cassette,by inserting protein coding sequence into the loop of a dsRNA.

In another embodiment, a method of the present invention for obtaining anucleic acid fragment comprising a nucleotide sequence for producing asubstantial portion of a dsRNA or siRNA comprises: (a) synthesizing afirst and a second oligonucleotide primers corresponding to a portion ofone of the nucleotide sequences from a targeted pathogen; and (b)amplifying a nucleic acid insert present in a cloning vector using thefirst and second oligonucleotide primers of step (a) wherein theamplified nucleic acid molecule transcribes a substantial portion of thea substantial portion of a dsRNA or siRNA of the present invention.

In practicing the present invention, target genes may be derived from abegomovirus, tospovirus, or potexvirus. It is contemplated that severalcriteria may be employed in the selection of preferred target genes.Such sequences may be identified by aligning, for instance, begomovirusor tospovirus sequences from multiple strains and/or species. Abioinformatics approach has identified numerous 21-mers that are mostlyconserved in more than 100 begomovirus strains and species. In someinstances, mismatches within a particular 21-mer are allowed for broadertargeting.

As used herein, the term “derived from” refers to a specified nucleotidesequence that may be obtained from a particular specified source orspecies, albeit not necessarily directly from that specified source orspecies.

In one embodiment, a gene is selected that results in suppression ofviral replication and/or symptomatology. Other target genes for use inthe present invention may include, for example, those that playimportant roles in viral transmission, movement within a plant, orvirion assembly (e.g. tospovirus terminal sequences, comprising theterminal repeat sequences). According to one aspect of the presentinvention for virus control, the target sequences may essentially bederived from the targeted plant viruses. Some of the exemplary targetsequences cloned from a begomovirus, tospovirus, or potexvirus may befound in the Sequence Listing, for instance in SEQ ID NOs: 1, 7, 13, 19,25, 31, 37, 43, 47, 51, 55, 59, 63, 67, 71, 85, 99, 113, 127, and 141,as well as within SEQ ID NOs:169-455.

For the purpose of the present invention, the dsRNA molecules may beobtained by polymerase chain (PCR) amplification of a target genesequences derived from a gDNA or cDNA library or portions thereof. TheDNA library may be prepared using methods known to the ordinary skilledin the art and DNA/RNA may be extracted. Genomic DNA or cDNA librariesgenerated from a target organism may be used for PCR amplification forproduction of the dsRNA or siRNA.

The target gene sequences may be then be PCR amplified and sequencedusing the methods readily available in the art. One skilled in the artmay be able to modify the PCR conditions to ensure optimal PCR productformation. The confirmed PCR product may be used as a template for invitro transcription to generate sense and antisense RNA with theincluded minimal promoters. In one embodiment, the present inventioncomprises isolated and purified nucleotide sequences that may be used asplant-virus control agents. The isolated and purified nucleotidesequences may comprise those as set forth in the sequence listing.

As used herein, the phrase “coding sequence,” “structural nucleotidesequence” or “structural nucleic acid molecule” refers to a nucleotidesequence that is translated into a polypeptide, usually via mRNA, whenplaced under the control of appropriate regulatory sequences. Theboundaries of the coding sequence are determined by a translation startcodon at the 5′-terminus and a translation stop codon at the3′-terminus. A coding sequence can include, but is not limited to,genomic DNA, cDNA, EST and recombinant nucleotide sequences.

The term “recombinant DNA” or “recombinant nucleotide sequence” refersto DNA that contains a genetically engineered modification throughmanipulation via mutagenesis, restriction enzymes, and the like.

EXAMPLES

The inventors herein have identified a means for controlling virusinfections in plants by incorporating into plants engineered miRNAs,ta-siRNAs and/or phased siRNAs. Any one or any combination of theseattributes can result in an effective inhibition of plant infection,inhibition of plant disease, and/or reduction in severity of diseasesymptoms.

Example 1 Targets for Multivirus Resistance

Sequences of targeted viruses were assembled from Genbank. FIG. 1schematically shows the genome organization of representative targetedviruses. For targeted begomoviruses, 58 isolates of Tomato yellow leafcurl virus (TYLCV), 30 isolates of Tomato leaf curl New Delhi virus(ToLCNDV), 5 isolates of Tomato severe leaf curl virus (ToSLCV), 5isolates of Pepper golden mosaic virus (PepGMV), and 2 isolates ofPepper huasteco yellow vein virus (PHYVV) were analyzed. For targetedtospoviruses, Tomato spotted wilt virus (TSWV), Groundnut bud necrosisvirus (GBNV) and Capsicum chlorosis virus (CaCV), 6 L segments (TSWV: 4;GBNV: 1; CaCV: 1), 23 M segments (TSWV: 20; GBNV: 2; CaCV: 1), and 45 Ssegments (TSWV: 41; GBNV: 2; CaCV: 2) were analyzed. For isolates of thetargeted potexvirus, 13 isolates of Pepino mosaic virus (PepMV) wereanalyzed (e.g. Lopez et al., 2005; Cotillon et al., 2002). Abioinformatics approach was utilized to identify approximately 20-24nucleotide sequences to be used as artificial miRNAs to suppress as manyof the targeted viruses as possible.

The following Table (Table 1) lists sequences analyzed for possible usein designing transgene constructs for generating engineered miRNAs, aswell as for identifying sequences for use in dsRNA-mediated viralresistance approaches:

TABLE 1 Exemplary sequences used for bioinformatics analysis (SEQ IDNOs: 169-362) Virus type Virus name GenBank Accession Begomovirus Tomatoyellow leaf curl virus (TYLCV) AF024715, X15656, X76319, AB014346,AB014347, AB110217, AB110218, AB116629, AB116630, AB116631, AB116632,AB116633, AB116634, AB116635, AB116636, AF071228, AF105975, AF271234,AJ132711, AJ223505, AJ489258, AJ519441, AJ812277, AJ865337, AM282874,AM409201, AM698117, AM698118, AM698119, AY044138, AY134494, AY227892,AY502934, AY530931, AY594174, AY594175. DQ144621, DQ358913, DQ631892,DQ644565, EF051116, EF054893, EF054894, EF060196, EF101929, EF107520,EF110890, EF158044, EF185318, EF210554, EF210555, EF433426, EF523478,EF539831, EU031444, EU143745 Begomovirus Tomato leaf curl New Delhivirus (ToLCNDV) NC_004611, U15015, U15016, Y16421, AB330079, AB368447,AB368448, AF102276, AF448058, AF448059, AJ620187, AJ875157, AM286433,AM286434, AM292302, AM850115, AY286316, AY428769, AY939926, DQ116880,DQ116883, DQ116885, DQ169056, EF035482, EF043230, EF043231, EF063145,EF068246, EF450316, EF620534, EU309045 Begomovirus Tomato severe leafcurl virus (ToSLCV) AF130415, AJ508784, AJ508785, DQ347946, DQ347947Begomoivirus Pepper Huasteco yellow vein virus (PHYVV) NC_001359,X70418, AY044162 Begomovirus Pepper golden mosaic virus (PepGMV)NC_004101, U57457, AF149227, AY928512, AY928514, AY928516 TospovirusTomato spotted wilt wirus (TSWV) (L segment) NC_002052, D10066,AB190813, AB198742, AY070218 Tospovirus Tomato spotted wilt wirus (TSWV)(M segment) NC_002050, S48091, AB010996, AB190818, AF208497, AF208498,AY744481, AY744482, AY744483, AY744484, AY744485, AY744486, AY744487,AY744488, AY744489, AY744490, AY744491, AY744492, AY744493, AY870389,AY870390 Tospovirus Tomato spotted wilt wirus (TSWV) (S segment)NC_002051, D00645, AB088385, AB190819, AF020659, AF020660, AJ418777,AJ418778, AJ418779, AJ418780, AJ418781, AY744468, AY744469, AY744470,AY744471, AY744472, AY744473, AY744474, AY744475, AY744476, AY744477,AY744478, AY744479, AY744480, AY870391, AY870392, DQ376177, DQ376178,DQ376179, DQ376180, DQ376181, DQ376182, DQ376183, DQ376184, DQ376185,DQ398945, DQ431237, DQ431238, DQ915946, DQ915947, DQ915948 TospovirusGroundnut bud necrosis virus (GBNV) NC_003614, AF025538 (L segment)Tospovirus Groundnut bud necrosis virus (GBNV) NC_003620, U42555,AY871097 (M segment) Tospovirus Groundnut bud necrosis virus (GBNV)NC_003619, U27809, AY871098 (S segment) Tospovirus Capsicum chlorosisvirus (CaCV) (L segment) NC_008302, DQ256124 Tospovirus Capsicumchlorosis virus (CaCV) (M segment) DQ256125 Tospovirus Capsicumchlorosis virus (CaCV) (S segment) DQ355974, DQ256123 Potexvirus Pepinomosaic virus (PepMV) EF599605, AY509926, AJ438767, DQ000984, DQ000985,AY509927, EF408821, AM491606, AJ606360, AJ606359, AJ606361, AF484251,AM109896

Example 2 Virus Segments for RNAi

Selected viral genomes were divided to ˜350-500 bp fragments, partiallyoverlapping by about 50 bp (e.g. FIG. 2A-2D). Efficacy data wascollected for the ability of single sequences to control particularvirus species when expressed individually in transformed plants. Ininitial studies, segments corresponding to approximately 2.3 kB out ofthe 2.7 kB geminivirus DNA-A genome were screened (FIG. 2B; segments1-8). Likewise, as shown in FIG. 2C, segments 1-8 representingapproximately 4 kB out of the 6.5 kB PepMV (potexvirus) genome weretested. FIG. 2D shows the portions of the tripartite tospovirus genomethat were tested for efficacy: approximately 1.5 kB out of the 8.9 kB LvRNA (i.e. virus RNA) (segments 1-3); approximately 1.5 kB of the 5.4 kBM vRNA (segments 4-6); and approximately 1 kB of the 2.9 kB S vRNA(segments 7-8).

In one representative study, a nucleic acid segment corresponding to apart of the TSWV coat protein (nucleocapsid) was tested for efficacywhen expressed as an inverted repeat (FIG. 3). Virus resistance wasfound to correlate with siRNA production in tomato plants. Also in theTYLCV resistance screen, R₁ plants were first examined for the presenceof the transgene and then infected with the TYLCV infectious clone. As acontrol, a non-transformed (wild-type) tomato line was included; thesewild-type plants typically have less than 5% “resistance”, i.e. lessthan 5% of plants escape infection by inoculation in this assay. Incontrast, among CP4 positive plants (containing the selectable markergene specifying resistance to glyphosate, which is linked to theexpression cassette with viral sequence), 56% of R₁ plants comprising anintroduced nucleic acid segment targeting the replication protein (3constructs), 27% comprising an introduced nucleic acid segment targetingthe coat protein (3 constructs), and 38% comprising an introducednucleic acid segment targeting the rest of the coding regions (2construct) showed resistance to TYLCV.

Additional studies scanning these and additional portions of thetospovirus genome were then undertaken, in order to identify and defineportions of the viral genome which can mediate dsRNA-related virusresistance to representative viruses. These sequences were tested asinverted repeat segments in double stranded (dsRNA)-generatingconstructs, wherein tested constructs comprised a promoter operablylinked to a given sequence in an antisense orientation (e.g. SEQ IDNOs:379-442 as described below), followed by a loop sequence, and thenthe given sequence in a sense orientation, and a transcriptionalterminator. After transcription and base pairing of the inverted repeatsequences, the double stranded RNA regions are cleaved by a Dicer orDicer-like RNAse to generate the specific antiviral siRNAs which targetand interfere with viral gene expression.

Capsicum chlorosis virus (CaCV), Groundnut bud necrosis virus (GBNV),and Tomato spotted wilt virus (TSWV), among other tospoviruses. Virusresistance tests of transgenic R₁ plants demonstrated that all regionsof the tospovirus genome are equally as effective as the target ofdsRNA. Thus, significant virus resistance was seen following expressionof transcripts which disrupt expression of tospovirus coat protein (CP),as well as the glycoprotein-encoding virus genome segments GP1, GP2,GP3, and RNA-dependent-RNA polymerase (RdRP) protein segments. FIG.11A-B describes resistance results seen from transgenic plants. Witheach bar representing an individual transgenic event and target region,for instance 50-80% of R₁ plants expressing a dsRNA targeting the CPregion were resistant to CaCV, while ˜25-90% of R₁ plants expressing adsRNA targeting a virus glycoprotein showed virus resistance, and about25-80% of R₁ plants expressing a dsRNA targeting a virus RdRP showedresistance (e.g. FIG. 11B). dsRNA-mediated resistance to TSWV wasparticularly effective, with 100% of R₁ plants displaying virusresistance from all tested events with constructs targeting the coatprotein (FIG. 11A). Representative TSWV, CaCV, and GBNV sequencesselected for use in generation of dsRNAs targeting tospovirus geneexpression are listed in SEQ ID NOs:419-436 (in antisense orientation aslisted).

Additional studies demonstrated that all regions of the PepMV genome areequally effective in generating dsRNA-mediated resistance against thispotexvirus. FIG. 12 shows that in most cases, 95-100% of all transgenicR₁ plants comprising virus-encoded segments targeting CP (coat protein),CP/MOV (Coat Protein and Movement Protein sequences), RdRP, TGB (TripleGene Block motif protein implicated in viral cell-to-cell and longdistance movement in a host plant), or TGB/RdRP were resistant to PepMV.Representative PepMV sequences selected for use in generation of dsRNAstargeting potexvirus gene expression are listed in SEQ ID NOs:437-442,in antisense orientation as listed.

Likewise, regions of the begomovirus (geminivirus) genome were testedfor their ability to generate dsRNA-mediated resistance against thisvirus group. TYLCV, ToSLCV, PepGMV, PHYVV, and ToLCNDV were tested inbioassays as representative begomoviruses. FIGS. 13A-B shows that up toabout 70% of transgenic R₁ plants displayed resistance to a given viruswhen CP was the dsRNA target, while up to about 100% of transgenic R₁plants displayed resistance when a nucleic acid segment encoding viralreplication protein was the dsRNA target. Representative tospovirussequences selected for use in generation of dsRNAs targeting begomovirusgene expression are listed in SEQ ID NOs:379-418, in antisenseorientation as listed.

Together, numerous effective dsRNA targets for viruses in these threegenera (i.e. tospovirus, potexvirus, begomovirusl ) were identified.

Based on these RNAi results, sequences targeting efficacious targetregions were selected for each virus and fused into two transgeniccassettes on a nucleic acid construct, to target multiple viruses inmultiple virus families. An example of this approach is shown in FIG.14. For begomoviruses, a Rep region was used, while for tospovirus andpotexvirus, CP regions were selected since the CP region is relativelymore conserved among different strains of the same viruses. Some of thesequences utilized in the cassettes were further modified, in view ofexpected G::U base pairings, to allow for broadening of protection torelated viral strains. Representative sequences selected for use ingeneration of dsRNAs targeting virus gene expression in the cassettesare listed in SEQ ID NOs:443-446 for “cassette 1” of FIG. 14, and in SEQID NOs:447-451 for “cassette 2” of FIG. 14, each in antisenseorientation as listed in the sequence listing. Thus, for instance, thenucleic acid segment targeting GBNV-CP1 which is found in cassette 1(SEQ ID NO:443) is similar to the segment targeting GBNV-CP1 as found inSEQ ID NO:429, but may have modification as noted above, and likewisefor the other nucleic acid segments used in the cassettes, relative tothe target segments as initially tested for relative efficacy.

Example 3 Artificial dsRNA Fusion Constructs for Multivirus Resistance

Nucleotide segments for dsRNA expression, from two or three of thedifferent virus groups (geminivirus, tospovirus, potexvirus), were alsocombined in a single expression cassette and plants were generated fromtransformed cells and analyzed for resistance to more than one virus.This was accomplished by fusing viral genomic fragments that testedeffective for generating siRNAs conferring virus resistance, forinstance as shown in Example 2. Analogously to Example 2, thesesequences were tested as inverted repeat segments in double stranded(dsRNA)-generating constructs, wherein tested constructs comprised apromoter operably linked to a given sequence in an antisense orientation(e.g. SEQ ID NOs:452-454 as described below), followed by a loopsequence, and then the given sequence in a sense orientation, and atranscriptional terminator. After transcription and base pairing of theinverted repeat sequences, the double stranded RNA regions are cleavedby a Dicer or Dicer-like RNAse to generate the specific antiviral siRNAswhich target and interfere with viral gene expression. In this approach,specific dsRNA segments for each of the targeted virus were used formultiple virus resistance (FIG. 4A).

Alternatively, conserved regions of the various viral genomes mayprovide a broader spectrum of resistance against different variantswithin the species or closely related species. Additionally, invertedrepeats comprising sequences that are highly similar to, but less than100% identical to, targeted segments of viral genomes may be designedand tested for efficacy. This approach, i.e. partially similar or“imperfect” inverted repeats, may broaden RNA-mediated protection torelated virus strains and species. For instance, since G-U basepairingat the RNA level has a comparable degree of thermodynamic stability assome typical Watson-Crick baseparing (e.g. A-U), some imperfect repeatsmay nonetheless activate RISC and guide cleavage of both perfect andimperfect viral targets.

In this instance, such partially similar inverted repeats may alsocomprise one or more sub-sequences of at least 21 nucleotides in lengththat display near 100% identity to one or more viral target sequences,in order to stimulate RNAi-mediated virus resistance. Thus for instance,the overall identity of an imperfect repeat segment to one or more viraltarget(s) may be as low as 75%, but with 2-4 or more sub-sequences of21-24 nucleotides that display 100% identity to a viral target sequence.Additionally, the presence of such “imperfect” dsRNA may help toincrease accumulation of dsRNA in plants and prevent the triggering oftranscriptional gene silencing. Thus, nucleotide segments from more thanone strain of a virus, or from more than one virus, may be utilized indesign and preparation of a fusion construct for dsRNA expression.

Such sequences may be identified by aligning, for instance, geminivirusor tospovirus sequences from multiple strains and/or species. Table 2shows percent similarities between geminivirus targets at the wholegenome level. Table 3 shows percent similarities between tospovirustargets at the whole genome level.

TABLE 2 Percent similarities between geminivirus targets (whole genome).TYLCV ToLCNDV PHYVV ToSLCV PepGMV TYLCV — 71 64 63 60 ToLCNDV 71 — 62 6059 PHYVV 64 62 — ToSLCV 63 60 67 — 76 PepGMV 60 59 66 76 —

TABLE 3 Percent similarities between tospovirus targets (whole genome).Segment Virus CapCV GBNV TSWV L vRNA CapCV_L — 44 45 8.8 kb GBNV_L 44 —58 TSWV_L 45 58 — M vRNA CapCV_M — 78 53 4.8-5.4 kB GBNV_M 78 — 54TSWV_M 53 54 — S vRNA CapCV_S — 65 42 2.9-3.3 kB GBNV_S 73 — 48 TSWV_S49 49 —

FIG. 4B schematically illustrates exemplary fusion constructs for dsRNAexpression. Coding sequences, including sequences lacking significantidentity with human and host plant genomes, were selected for generationof inverted repeats. Viral-derived segments may be oriented, forinstance, “antisense-loop-sense” for dsRNA expression. This approach canreduce the size of a transgenic dsRNA cassette required for multiplevirus resistance. The schematically described artificial fusionconstructs of FIG. 4B are listed as SEQ ID NOs: 452-453. SEQ ID NO:452artificial coat protein construct comprises sequences as follows: by1-157: TYLCV precoat protein for the loop; by 158-507 (i.e. 350 bpsegment) targeting TYLCV and ToLCNDV CP; by 508-851 (i.e. 344 bpsegment) targeting ToSLCV, PepGMV, and PHYVV CP expression. SEQ IDNO:453 comprises segments as follows: by 1-160 targeting TSWV coatprotein (CP) for the loop; by 161-456 (i.e. 296 bp segment) targetingTSWV CP; by 457-687 (i.e. 231 bp segment) targeting the PepMV CP; by688-919 (i.e. 232 bp segment) targeting the CaCV and GBNV CP. Efficacyresults for SEQ ID NO:453, the artificial CP sequence against multipletospoviruses and PepMV, are shown in FIG. 15. An additional artificialdsRNA fusion construct targeting Rep proteins of five geminiviruses(TYLCV, ToLCNDV, ToSLCV, PepGMV, and PHYVV) is given as SEQ ID NO:454.In this sequence, by 1-150 target TYLCV Rep protein for the loop; by151-500 (i.e. 350 bp segment) target TYLCV and ToLCNDV Rep protein; by501-860 (i.e. 360 bp segment) target ToSLCV, PepGMV, and PHYVV Repprotein.

Example 4 Engineered miRNA Approach: Selection of Suitable Sequences

The bioinformatics approach described in Example 1 was used to identifyseveral suitable sequences, approximately 21 nt in length, that targetgeminivirus sequences thought to be useful for miRNA-mediatedsuppression of geminivirus replication and/or symptom expression (Table4; FIG. 5).

TABLE 4 Suitable 21 nt sequences against targeted Geminiviruses. SEQmiRNA Sequence ID NO: Name (5′ → 3′) Target 1 Gemini 1TGTCATCAATGACGTTGTACT Rep 7 Gemini 2 TGGACTTTACATGGGCCTTCA Coat Protein13 Gemini 3 TACATGCCATATACAATAGCA Coat Protein 19 Gemini 4TCATAGAAGTAGATCCGGATT Coat Protein 25 Gemini 5 TTCCCCTGTGCGTGAATCCGTC2/C3 31 Gemini 6 TTCCGCCTTTAATTTGGATTG Rep 37 Gemini 7TTACATGGGCCTTCACAGCCT Coat Protein

The bioinformatics approach described in Example 1 was used to identifyseveral suitable sequences, approximately 21 nt in length, that targetTospovirus sequences thought to be useful for miRNA-mediated suppressionof Tospovirus replication and/or symptom expression (Table 5; FIG. 6).

TABLE 5 Suitable 21 nt sequence for each genomicsegment against three targeted Tospoviruses. SEQ ID miRNA Sequence NO:Name (5′ → 3′) Target 43 TospoL1-1  TTTAGGCATCATATAGATAGCT RdRP 47Tospo L1-2 TGATTTAGGCATCATATAGAT RdRP 51 Tospo M1 TATCTATATTTTCCATCTACCGP 55 Tospo M2 TTAGTTTGCAGGCTTCAATTA NSm 59 Tospo M3TTGCATGCTTCAATGAGAGCT NSm 63 Tospo S2 TTGACGTTAGACATGGTGTTT N 67Tospo S3 TAGAAAGTTTTGAAGTTGAAT N

The bioinformatics approach described in Example 1 was used to identifyseveral suitable sequences, approximately 21 nt in length, that targetpotexvirus sequences thought to be useful for miRNA-mediated suppressionof Tospovirus replication and/or symptom expression (Table 6; FIG.7A-7B).

TABLE 6 Suitable ~21 nt sequences against targeted potexvirus. SEQ IDmiRNA Sequence NO: Name (5′ → 3′) Target 71 PepMV1 TCTTCATTGTAGTTAATGGAGRdPR 85 PepMV2 TTGGAAGAGGAAAAGGTGGTT RdPR 99 Pep MV3TCAATCATGCACCTCCAGTCG RdPR 113 PepMV4 TAAGTAGCAAGGCCTAGGTGA TGBp1 127PepMV5 TTTGGAAGTAAATGCAGGCTG TGBp2 141 PepMV6  TAACCCGTTCCAAGGGGAGAAGTGBp3

Example 5 Incorporation of miRNA-Generating Sequences in a MIR GeneBackbone

The miRNA-generating sequences of Tables 4-6 were incorporated intotransgene constructs by gene synthesis. For instance, the wildtype MIRgene backbone “MON1” (see U.S. Publication 2007/0300329) from soybeanwith the wildtype 21 nt miRNA-generating sequence (SEQ ID NO:155 of thepresent application) was altered to replace the wildtypemiRNA-generating sequence with the Gemini1 target (SEQ ID NO:1),resulting in the Gemini1/MON1 construct (SEQ ID NO:156). Thus, a MIRbackbone (e.g. the MON1 backbone of SEQ ID NO:155) can be altered byreplacing the wildtype 21 nt miRNA-generating sequence with any of themiRNA sequences of Tables 4-6.

Similarly, MONS from soybean (SEQ ID NO:157), MON13 from rice (SEQ IDNO:159), MON18 from maize (SEQ ID NO:161), miR159a from maize (SEQ IDNO:163), or miR167g (SEQ ID NO:165), were used as backbone sequenceswith miRNA-generating sequences of Tables 4-6. Thus, the followingsequences were created (Table 7):

TABLE 7 Exemplary sequences incorporated into MIR gene backbone.miRNA-generating sequence MIR backbone Incorporated construct (SEQ IDNO) (SEQ ID NO) (SEQ ID NO) Gemini 1 MON1 Gemini1/MON1 (SEQ ID NO: 1)(SEQ ID NO: 155) (SEQ ID NO: 156) Gemini 4 MON5 Gemini 4/MON5 (SEQ IDNO: 19) (SEQ ID NO: 157) (SEQ ID NO: 158) Gemini 7 MON13 Gemini 7/MON13(SEQ ID NO: 37) (SEQ ID NO: 159) (SEQ ID NO: 160) PepMV5 MON18PepMV5/MON18 (SEQ ID NO: 127) (SEQ ID NO: 161) (SEQ ID NO: 162) Gemini 6miR159a Gemini 6/miR159a (SEQ ID NO: 31) (SEQ ID NO: 163) (SEQ ID NO:164) PepMV6 miR167g PepMV6/miR167g (SEQ ID NO: 141) (SEQ ID NO: 165)(SEQ ID NO: 166) Gemini 2 MON13 Gemini 2/MON13 (SEQ ID NO: 7) (SEQ IDNO: 159) (SEQ ID NO: 367) Gemini 3 MON1 Gemini3/MON1 (SEQ ID NO: 13)(SEQ ID NO: 155) (SEQ ID NO: 368) Gemini 5 miR159a Gemini 5/MiR159a (SEQID NO: 25) (SEQ ID NO: 163) (SEQ ID NO: 369) PepMV1 miR159aPepMV1/miR159a (SEQ ID NO: 71) (SEQ ID NO: 163) (SEQ ID NO: 363) PepMV2MON5 PepMV2/MON5 (SEQ ID NO: 85) (SEQ ID NO: 157) (SEQ ID NO: 364)PepMV3 MON13 PepMV3/MON13 (SEQ ID NO: 99) (SEQ ID NO: 159) (SEQ ID NO:365) PepMV4 MON1 PepMV4/MON1 (SEQ ID NO: 113) (SEQ ID NO: 155) (SEQ IDNO: 366) TospoL1-1 miR167g TospoL1-1/miR167g (SEQ ID NO: 43) (SEQ ID NO:165) (SEQ ID NO: 370) TospoL1-2 MON1 TospoL1-2 /MON1 (SEQ ID NO: 47)(SEQ ID NO: 155) (SEQ ID NO: 371) TospoM2 MON1 TospoM2/MON1 (SEQ ID NO:55) (SEQ ID NO: 155) (SEQ ID NO: 372) TospoM3 MON13 TospoM3/MON13 (SEQID NO: 59) (SEQ ID NO: 159) (SEQ ID NO: 373) TospoS2 MON5 TospoS2/MON5(SEQ ID NO: 63) (SEQ ID NO: 157) (SEQ ID NO: 374) TospoS3 MON13TospoS3/MON13 (SEQ ID NO: 67) (SEQ ID NO: 159) (SEQ ID NO: 375)Other selected miRNA-generating sequences, for instance from among anyof SEQ ID NOs:1-154, or from within SEQ ID NOs:169-362 or portionsthereof, may be utilized with MIR backbone sequences, for instance MON1from soybean, MONS from soybean, MON13 from rice, MON18 from maize,miR159a from maize, or mi167g from maize (SEQ ID NOs:155, 157, 159, 161,163, or 165) to create additional efficacious miRNA-generatingconstructs. Additionally, the MIR backbone sequences may be modified forinstance by replacing the portion of the sequence of the plant-derivedbackbone sequence which specifies an miRNA with a selected virus-derivedor artificial (e.g. consensus) sequence, and/or shortened, e.g. adeletion made at the 3′ and/or 5′ end, for instance as reflected in themiRNA-generating sequences and MIR backbone portions of SEQ ID NOs:158,160, 162, 164, 166, 363, 364, 365, 367, 369, 370, 373, 374, and 375,described below.

21-mers identified from within SEQ ID NOs:169-362, for instance among orwithin SEQ ID NOs:1-154, are tested individually, or two or more at atime while present in one expression cassette, for efficacy intransgenic tomato plants against appropriate viruses such as TYLCV andTSWV. Multiple effective 21-mers are expressed in one transgeniccassette using a polycistronic or a phased siRNA backbone (e.g. FIG. 8).Results of virus resistance assays from such testing of 21-mers aregiven in Example 7.

Once the efficacious antiviral miRNAs are identified, multiple miRNAsare deployed by expressing as a single transgenic trait, to achievemultiple virus resistance in transgenic plants. This is accomplished byfusing engineered MIR gene(s) described above together (e.g. to createSEQ ID NOs:156, 158, 160, 162, 164, 166, 363-375), or using a ta-siRNAor a phased siRNA gene structure that can deliver multiple antiviral ˜21nt sequences (e.g. FIG. 8). The later approaches are accomplished via anew round of gene synthesis to replace wild-type siRNAs with theefficacious antiviral 21 nt sequences in the ta-siRNA or phased siRNAbackbone.

Example 6 Supplementing RNA-Based Resistance

The RNA-mediated resistance approach utilizing dsRNA or miRNA can besupplemented by placing antiviral sequences, e.g. ones that encode aprotein, into the loop of a dsRNA-encoding sequence, or by inserting asequence that encodes an efficacious dsRNA or miRNA into an intron of apolypeptide expression cassette (intronic dsRNA/miRNA; Frizzi et al.,2008) (e.g. FIG. 9). For example, viral protein(s) such as coat proteinand/or replicase may be expressed in a transgenic plant that alsoexpresses an efficacious dsRNA, miRNA, or siRNA, without use of anadditional transgene cassette, by inserting protein coding sequence intothe loop of a dsRNA. A sequence that encodes a peptide aptamer thatinterferes with geminivirus replication may also be employed (e.g.Lopez-Ochoa et al., 2006).

Thus, artificial sequences may be expressed along with the dsRNA, miRNA,or siRNA in order to augment the virus resistant phenotype. Forinstance, an artificial nucleotide loop engineered from the tospovirusgenome may be used. The tospovirus (ssRNA) genome comprises a“panhandle” structure due to the presence of conserved terminal repeatsof 8-11 nt at both ends of each genome component. These terminal repeatsfrom each component of the genome (SEQ ID NOs:167-168), for instancefrom GBNV (Groundnut bud necrosis virus) or CaCV (Capsicum chlorosisvirus), may be fused and used as part of a loop-forming sequence in aplant transformation construct. Such an artificial sequence maycomprise, for instance, SEQ ID NOs:376-378, or SEQ ID NO:455, and canserve as an artificial substrate competing for reverse transcriptase,may interfere with proper circularization of replicating viral genomecomponents, may themselves generate nucleotide segments efficacious viaRNAi, or one or more of the above, thus interfering with virionassembly.

FIG. 16 demonstrates that inclusion of tospovirus terminal sequences ina construct for generating dsRNA results in measurable resistance to avirus such as CaCV. In the tested construct, SEQ ID NOs:376-378,themselves comprising SEQ ID NOs:168, were fused to create SEQ ID NO:455(i.e. by 1-247 of SEQ ID NO:455 comprise SEQ ID NO:376; by 248-303comprise SEQ ID NO:377, and by 304-369 comprise SEQ ID NO:378). Theconstruct was tested in both sense and antisense orientations, and thelevel of virus resistance was compared to that demonstrated by a controltomato plant, non-transgenic but otherwise isogenic.

Sequences encoding, for instance, coat protein or replicase, as well asartificial sequences described above, may be embedded within an intronsequence as well. Thus, multiple modes of action may be deployed bybeing engineered into a single expression cassette (e.g. FIG. 9), ormore than one expression cassette.

Example 7 Results of Exemplary Virus Resistance Assays Using EngineeredmiRNAs

Engineered miRNA-producing constructs as listed in Table 7 were testedfor efficacy.

Bioassay results for geminivirus and potexvirus assays are shown inTables 8-9. Virus resistance was observed due to miRNA expression, andcorrelating to expression and proper processing of a given transgenetranscript. For instance, for SEQ ID NO:166, proper processing ofPepMV6/mir167g was not observed, nor was virus resistance seen for thisconstruct, thus correlating miRNA production with virus resistance intransgenic R₁ plants. Regarding tospovirus experiments, expression andproper processing of transcripts from SEQ ID NOs:370, 371, 373, and 375was observed, targeting, respectively, the RdRP, RdRP, NsM, and N genes.CP4-positive R₁ plants displayed reduced symptoms when infected withTSWV. For SEQ ID NOs:372 and 374, targeting, respectively, the NsM and Ngenes, proper processing of miRNA was not observed, and no reduction insymptoms was noted.

The synthetic miRNAs were most effective against PepMV, and gave delayedor reduced virus infection symptoms against geminiviruses andtospovirus. The constructs can be combined in stacked miRNA cassettes inorder to synergistically inhibit the target viruses. Since multiplemiRNAs can be expressed from a single expression cassette, constructsexpressing, for instance, 8 or 10 antiviral miRNAs can be created (asshown in FIG. 10A-10B). As an additional approach, antiviral miRNAexpression cassettes can be inserted as the “loop” sequence (e.g. FIG.9) of a dsRNA expression cassette, to combine the dsRNA and miRNAmechanisms for viral control (see FIG. 10C).

TABLE 8 Results of geminivirus resistance assays utilizing engineeredsequences incorporated into MIR gene backbones. Incorporated construct(SEQ % Virus Resistance^(b) Target ID NO) miRNA Expression^(a) TYLCVToLCNDV PepGMV PHYVV Region SEQ ID NO: 156 Gemini1/ yes 16 ± 16% 31 ± 1737 ± 13 29 ± 16 Rep MON1 SEQ ID NO: 367 Gemini 2/ yes 34 ± 27 27 ± 14 29± 4 32 ± 16 CP MON13 SEQ ID NO: 368 Gemini3/ yes 13 ± 8 23 ± 7 34 ± 1114 ± 19 CP MON1 SEQ ID NO: 158 Gemini4/ no — — — — CP MON5 SEQ ID NO:369 Gemini5/ yes 14 ± 19 30 ± 20 51 ± 16 28 ± 37 C2/C3 miR159a SEQ IDNO: 164 Gemini6/ yes 20 ± 13 23 ± 12 36 ± 10 23 ± 19 Rep miR159a SEQ IDNO: 160 Gemini 7-1/ yes 7 28 40 11 CP MON13 ^(a)proper processing of themiRNA detected in transgenic plants ^(b)average percentage ± s.d. of R1CP4-positive segregants for resistance to tomato yellow leaf curl virus(TYLCV), Tomato leaf curl New Delhi virus (ToLCNDV), Pepper goldenmosaic virus (PePGMV), or Pepper Huasteco yellow vein virus (PHYVV.

TABLE 9 Results of Pepino mosaic virus (PepMV) resistance assaysutilizing engineered sequences incorporated into MIR gene backbones.Incorporated construct % Virus Target (SEQ ID NO) miRNA Expression^(a)Resistance^(b) Region 363 PepMV1/miR159a yes Not tested RdRP 364PepMV2/MON5 yes (weak) 10 ± 14 RdRP 365 PepMV3/MON13 yes 100 ± 0  RdRP366 PepMV4/MON1 yes (weak) 44 ± 52 TGBp1 162 PepMV5/MON18 yes 99 ± 3 TGBp2 166 PepMV6/miR167g no — TGBp3 ^(a)proper processing of the miRNAdetected in transgenic plants ^(b)average percentage ± s.d. of R1CP4-positive segregants for resistance to PepMV.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

REFERENCES

The references listed below are incorporated herein by reference to theextent that they supplement, explain, provide a background, or teachmethodology, techniques, and/or compositions employed herein.

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What is claimed is:
 1. A tomato plant comprising resistance to aplurality of plant virus species, wherein the resistance is provided byvirus-derived sequences with at least two different modes of actionselected from the group consisting of a dsRNA, miRNA being complementaryto all or part of a target gene of said virus species, and inhibition oftospovirus virion assembly by expression of a tospovirus genome segmentterminal sequence, further wherein resistance provided to at least oneof the plant virus species is provided by expression of a nucleic acidconstruct that produces miRNA, and wherein the viruses are selected fromthe genera potexvirus, tospovirus, and begomovirus.
 2. The plant ofclaim 1, wherein the resistance is provided by at least three differentmodes of action.
 3. The plant of claim 1, wherein the resistancecomprises resistance against a begomovirus, tospovirus or potexvirus. 4.The plant of claim 1, wherein resistance provided to at least one of theplant virus species is provided by expression of a nucleic acidconstruct that produces dsRNA.
 5. The plant of claim 4, whereinresistance provided to at least one of the plant virus species isprovided by expression of a dsRNA fusion construct.
 6. The plant ofclaim 4, wherein the dsRNA interferes with expression of a virus coatprotein gene, a virus movement protein gene or a virus replication gene.7. The plant of claim 4, wherein the nucleic acid construct whichproduces dsRNA comprises SEQ ID NO:444.
 8. The plant of claim 1, whereinresistance against a begomovirus or tospovirus is provided by a sequencecomprised within a stacked miRNA expression cassette.
 9. The plant ofclaim 1, wherein the miRNA interferes with expression of a virus coatprotein gene, a virus movement protein gene or a virus replication gene.10. The plant of claim 1, wherein the miRNA comprises SEQ ID NO:1. 11.The plant of claim 1, wherein: (a) resistance against a begomovirus isprovided by expression of a dsRNA which interferes with expression of abegomovirus replication gene; (b) resistance against a tospovirus orpotexvirus is provided by expression of a dsRNA which interferes withexpression of a virus coat protein gene or virus movement protein gene;(c) resistance against a potexvirus is provided by expression of anucleic acid construct which produces a miRNA; or (d) resistance againsta begomovirus or tospovirus is provided by a sequence comprised within astacked miRNA expression cassette.
 12. The plant of claim 1, whereinresistance provided to at least one of the plant virus species isprovided by expression of a tospovirus genome segment terminal sequencethat inhibits virion assembly.
 13. The plant of claim 1, whereinresistance provided to at least one of the plant virus species isprovided by inhibiting tospovirus virion assembly, wherein virionassembly is inhibited by a sequence comprised within a nucleic acidconstruct comprising a first nucleic acid segment and a second nucleicacid segment, wherein the first and second segments are substantiallyinverted repeats of each other and are linked together by a thirdnucleic acid segment, and wherein the third segment comprises at leastone terminal sequence of a tospovirus genome segment that inhibitsvirion assembly.
 14. The plant of claim 13, wherein the third nucleicacid comprises a tospovirus genome terminal sequence selected from thegroup consisting of: a terminal sequence of a CaCV or GBNV L genomesegment, a terminal sequence of a CaCV or GBNV M genome segment, aterminal sequence of a CaCV or GBNV S genome segment, a tospovirusgenome terminal repeat sequence, a nucleic acid sequence comprising SEQID NO: 167, a nucleic acid sequence comprising SEQ ID NO:168, a nucleicacid sequence comprising SEQ ID NO:376, a nucleic acid sequencecomprising SEQ ID NO:377, a nucleic acid sequence comprising SEQ IDNO:378, and a nucleic acid sequence comprising SEQ ID NO:
 455. 15. Theplant of claim 14, wherein the tospovirus genome segment terminal repeatsequence comprises SEQ ID NO:167 or SEQ ID NO:168.
 16. The plant ofclaim 1, wherein the viruses are selected from the group consisting of:a) at least one of TYLCV, ToSLCV, ToLCNDV, PHYVV, PepGMV; b) one or moreof TSWV, GBNV, CaCV; and c) PepMV.
 17. The plant of claim 1, wherein thepotexvirus is Pepino mosaic virus.
 18. The plant of claim 1, wherein thebegomovirus is TYLCV, ToLCNDV, PHYVV, ToSLCV, or PepGMV.
 19. The plantof claim 1, wherein the tospovirus is CaCV, GBNV, or TSWV.
 20. The plantof claim 1, wherein the begomovirus is TYLCV and the potexvirus isPepino mosaic virus; or the tospovirus is TSWV and the potexvirus isPepino mosaic virus; or the wherein the begomovirus is TYLCV, thepotexvirus is Pepino mosaic virus, and the tospovirus is TSWV.
 21. Theplant of claim 1, further comprising a sequence selected from the groupconsisting of SEQ ID NOs:156, 158, 160, 162, 164, 166, and 363-375. 22.The plant of claim 1, further defined as comprising SEQ ID NO:1.
 23. Theplant of claim 1, comprising (a) SEQ ID NO:444 and at least one sequenceselected from the group consisting of SEQ ID NOs:167, 168, 376, 377,378, and 455; (b) SEQ ID NO: 444 and SEQ ID NO:1; or (c) SEQ ID NO:1 andat least one sequence selected from the group consisting of SEQ IDNOs:167, 168, 376, 377, 378, and
 455. 24. The plant of claim 1, whereinthe plant comprises at least one heterologous nucleic acid sequence thatconfers viral resistance selected from the group consisting of a) anucleic acid sequence that encodes an RNA sequence that is complementaryto all or a part of a first target gene; b) a nucleic acid sequence thatcomprises multiple copies of at least one anti-sense DNA segment that isanti-sense to at least one segment of said at least one first targetgene; c) a nucleic acid sequence that comprises a sense DNA segment fromat least one target gene; d) a nucleic acid sequence that comprisesmultiple copies of at least one sense DNA segment of a target gene; e) anucleic acid sequence that transcribes to RNA for suppressing a targetgene by forming double-stranded RNA and that comprises at least onesegment that is anti-sense to all or a portion of the target gene and atleast one sense DNA segment that comprises a segment of said targetgene; f) a nucleic acid sequence that transcribes to RNA for suppressinga target gene by forming a single double-stranded RNA that comprisesmultiple serial anti-sense DNA segments that are anti-sense to at leastone segment of the target gene and multiple serial sense DNA segmentsthat comprise at least one segment of said target gene; g) a nucleicacid sequence that transcribes to RNA for suppressing a target gene byforming multiple double strands of RNA and comprises multiple segmentsthat are anti-sense to at least one segment of said target gene andmultiple sense DNA segments of the target gene, and wherein saidmultiple anti-sense DNA segments and said multiple sense DNA segmentsare arranged in a series of inverted repeats; h) a nucleic acid sequencethat comprises nucleotides derived from a plant miRNA; and i) a nucleicacid sequence encoding at least one tospovirus terminal sequence thatinterferes with virion assembly.
 25. The plant of claim 24, furthercomprising a non-transgenic plant virus resistance trait.
 26. The plantof claim 24, wherein expression of the at least one heterologous nucleicacid sequence results in resistance to two or more viruses selected fromthe group consisting of: tospoviruses, begomoviruses, and potexviruses.27. A transgenic seed of the plant of claim
 1. 28. A method forconferring resistance in a tomato plant to a plurality of plant virusspecies, the method comprising expressing in the plant at least twovirus-derived nucleic acid sequences that collectively provideresistance to said plurality of plant virus species, wherein at least 2different modes of action are utilized to provide such resistance,comprising expression of at least two sequences selected from the groupconsisting of: a dsRNA, miRNA being complementary to all or part of atarget gene of said virus species, and a sequence which interferes withtospovirus virion assembly by expression of a tospovirus genome segmentterminal sequence, further wherein resistance provided to at least oneof the plant virus species is provided by expression of a nucleic acidconstruct that produces miRNA, and wherein the viruses are selected fromthe genera potexvirus, tospovirus, and begomovirus.
 29. The method ofclaim 28, wherein resistance provided to at least one of the plant virusspecies is provided by expression of a nucleic acid construct thatproduces dsRNA.
 30. The method of claim 28, wherein resistance providedto at least one of the plant virus species is provided by expression ofa dsRNA fusion construct.
 31. The method of claim 29, wherein the dsRNAinterferes with expression of a virus coat protein gene, a virusmovement protein gene or a virus replication gene.
 32. The method ofclaim 29, wherein the nucleic acid construct comprises SEQ ID NO:444.33. The method of claim 28, wherein resistance against a begomovirus ortospovirus is provided by a sequence comprised within a stacked miRNAexpression cassette.
 34. The method of claim 28, wherein the miRNAinterferes with expression of a virus coat protein gene, a virusmovement protein gene or a virus replication gene.
 35. The method ofclaim 28, wherein the miRNA comprises SEQ ID NO:1.
 36. The method ofclaim 28, wherein: (a) resistance against a begomovirus is provided byexpression of dsRNA which interferes with expression of a begomovirusreplication gene; (b) resistance against a tospovirus or potexvirus isprovided by expression of a dsRNA which interferes with expression of avirus coat protein gene or virus movement protein gene; (c) resistanceagainst a potexvirus is provided by expression of a nucleic acidconstruct which produces miRNA; or (d) resistance against a begomovirusor tospovirus is provided by a sequence comprised within a stacked miRNAexpression cassette.
 37. The method of claim 28, wherein resistanceprovided to at least one of the plant virus species is provided byexpression of a tospovirus genome segment terminal sequence thatinhibits virion assembly.
 38. The method of claim 28, wherein resistanceprovided to at least one of said plant virus species is provided byinhibiting tospovirus virion assembly, wherein virion assembly isinhibited by a sequence comprised within a nucleic acid constructcomprising a first nucleic acid segment and a second nucleic acidsegment, wherein the first and second segments are substantiallyinverted repeats of each other and are linked together by a thirdnucleic acid segment, and wherein the third segment comprises at leastone terminal sequence of a tospovirus genome segment, expression ofwhich inhibits virion assembly.
 39. The method of claim 38, wherein thethird nucleic acid comprises a tospovirus genome terminal sequenceselected from the group consisting of: a terminal sequence of a CaCV orGBNV L genome segment, a terminal sequence of a CaCV or GBNV M genomesegment, a terminal sequence of a CaCV or GBNV S genome segment, and atospovirus genome terminal repeat sequence.
 40. The method of claim 39,wherein the terminal sequence or terminal repeat sequence comprises SEQID NO:167, SEQ ID NO:168, SEQ ID NO:376, SEQ ID NO:377, or SEQ ID NO:378.
 41. The method of claim 28, wherein the viruses are selected fromthe group consisting of: a) one or more of TYLCV, ToSLCV, ToLCNDV,PHYVV, PepGMV; b) one or more of TSWV, GBNV, CaCV; and c) PepMV.
 42. Themethod of claim 28, wherein the nucleic acid sequence comprises at leastone gene suppression element for suppressing at least one first targetgene.
 43. The method of claim 28, comprising expressing in the plant:(a) SEQ ID NO:444 and at least one sequence selected from the groupconsisting of SEQ ID NOs:167, 168, 376, 377, 378, and 455; (b) SEQ IDNO:444 and SEQ ID NO:1; or (c) SEQ ID NO:1 and at least one sequenceselected from the group consisting of SEQ ID NOs:167, 168, 376, 377,378, and
 455. 44. A transgenic plant cell of the plant of claim 1.